FAQ
1. iST technology for sample preparation
1.1. Is my own lysis buffer compatible for use with the PreOmics’ kits?
We recommend that you use the appropriate LYSE buffer provided with the PreOmics’ iST, iST-NHS or iST-BCT kits. If you want to use your own lysis buffer, please refer to the following table For all other buffers containing higher concentrations of salt, chaotropes, or detergents, we recommend using the SP3-iST Add-on kit in combination with the iST, iST-NHS, and iST-BCT kits (see FAQ 9.2. for more information on SP3-iST compatibility).
2X concentrated LYSE buffers have to be ordered in addition to the iST kits. Please contact us (info@preomics.com) for any further question on buffer compatibility or supply with reagents.
For urea-containing buffers: To reduce the risk of carbamylation of proteins, it is recommended not to heat samples above 37 °C in urea-containing buffers. Please contact us via info@preomics.com for recommendations on how to modify the protocol.
For urea-containing buffers: To reduce the risk of carbamylation of proteins, it is recommended not to heat samples above 37 °C in urea-containing buffers. Please contact us via info@preomics.com for recommendations on how to modify the protocol.
Several different protocols for protein precipitation exist. We recommend acetone precipitation:
1. Transfer protein lysate (not more than 300 µL) to a clean 2 mL microcentrifuge tube.
> in case you have a larger sample volume, use either a 5 or 15 mL tube
2. Add ice-cold acetone (-20°C) to your sample > add at least 4-fold more acetone than sample, e.g., 300 µL sample + 1.2 mL acetone
3. Mix briefly
4. Incubate for one hour at -20°C
5. Spin in table-top centrifuge at 4°C for 15 min at 13,000 rpm
6. Carefully discard supernatant, make sure not to disturb the pellet
7. Air-dry the pellet for 5-10 min8. Continue with appropriate PreOmics protocol adding the LYSE
> you can also freeze the pellet after air-drying at -20°C or -80°C until further use
1.2. What is the maximum starting volume of sample I can process with the PreOmics kits?
Sample volume (liquid or solid, e.g. cell pellets) should not exceed 10 µL when combined with the appropriate LYSE buffer. For sample volumes between 11-25 µL, use the 2-fold concentrated lysis buffers, which can be purchased upon request. The 2X lysis buffers are mixed 1:1 with samples ranging from 11-25 µL, e.g., 11 µL sample + 11 µL 2X LYSE, all downstream buffer volumes stay the same.
If your sample volume exceeds 25 µL, perform protein precipitation before processing with the standard PreOmics’ workflows.
If your sample volume exceeds 25 µL, perform protein precipitation before processing with the standard PreOmics’ workflows.
1.3. How do I perform protein precipitation?
Several different protocols for protein precipitation exist. We recommend acetone precipitation:
1. Transfer protein lysate (not more than 300 µL) to a clean 2 mL microcentrifuge tube.
> in case you have a larger sample volume, use either a 5 or 15 mL tube
2. Add ice-cold acetone (-20°C) to your sample > add at least 4-fold more acetone than sample, e.g., 300 µL sample + 1.2 mL acetone
3. Mix briefly
4. Incubate for one hour at -20°C
5. Spin in table-top centrifuge at 4°C for 15 min at 13,000 rpm
6. Carefully discard supernatant, make sure not to disturb the pellet
7. Air-dry the pellet for 5-10 min8. Continue with appropriate PreOmics protocol adding the LYSE
> you can also freeze the pellet after air-drying at -20°C or -80°C until further use
1. Transfer protein lysate (not more than 300 µL) to a clean 2 mL microcentrifuge tube.
> in case you have a larger sample volume, use either a 5 or 15 mL tube
2. Add ice-cold acetone (-20°C) to your sample > add at least 4-fold more acetone than sample, e.g., 300 µL sample + 1.2 mL acetone
3. Mix briefly
4. Incubate for one hour at -20°C
5. Spin in table-top centrifuge at 4°C for 15 min at 13,000 rpm
6. Carefully discard supernatant, make sure not to disturb the pellet
7. Air-dry the pellet for 5-10 min8. Continue with appropriate PreOmics protocol adding the LYSE
> you can also freeze the pellet after air-drying at -20°C or -80°C until further use
1.4. Can I add protease inhibitors to my sample?
Adding a protease inhibitor (cocktail) that inhibits serine proteases is not recommended when working with the iST, iST-BCT, or iST-NHS kit. This type of inhibitor reduces trypsin digestion efficiency, and the rate of missed cleavages will be significantly increased. This will result in a lower protein identification rate. Please also consult the safety data sheet of the protease inhibitor to check for possible impurities.
If a protease inhibitor (cocktail) is required for a specific application during the lysis process, we recommend using the SP3-iST workflow to remove the protease inhibitor prior to digestion.
If a protease inhibitor (cocktail) is required for a specific application during the lysis process, we recommend using the SP3-iST workflow to remove the protease inhibitor prior to digestion.
1.5. Can I use mechanical force to help lysis in combination with the PreOmics’ kits?
Many different mechanical force methods can be employed such as traditional bead milling, liquid nitrogen grinding, or commercial systems from various vendors (e.g., Bertin Instruments, Covaris, Hielscher, MP Biomedicals). These methods can aid lysis efficiency for samples including cells, yeast, or tissues. We recommend the PreOmics' BeatBox system which is easy to use and allows efficient and reproducible tissue homogenization and cell lysis with flexible throughput from 1-96 samples. See https://www.preomics.com/beatbox for more information.
Samples such as biological fluids do not require additional mechanical force disruption.
Samples such as biological fluids do not require additional mechanical force disruption.
1.6. What are the minimum and maximum protein amounts for the PreOmics’ kits?
Highly reproducible results are achieved with protein starting amounts ranging from 1 µg to 100 µg.
For low input information please see 1.10.
For low input information please see 1.10.
1.7. Which protein quantification methods are compatible with the PreOmics' kits?
To determine protein quantity in the sample lysed in PreOmics LYSE buffers, we recommend using the Pierce™ BCA Protein Assay Kit - Reducing Agent Compatible or the NanoOrange™ Protein Quantitation Kit (Thermo Fisher Scientific), depending on which LYSE is used. Below is the list of compatible LYSE buffers and the protein quantification assay with recommended dilution.
Protein quantification assays and compatible PreOmics LYSE buffers
Protein quantification assays and compatible PreOmics LYSE buffers
Assay
Pierce™ BCA Protein Assay Kit - Reducing Agent Compatible
Pierce™ Microplate BCA Protein Assay Kit - Reducing Agent Compatible
Pierce™ Microplate BCA Protein Assay Kit - Reducing Agent Compatible
NanoOrange™ Protein Quantitation Kit
Manufacturer
Thermo Fisher Scientific
Thermo Fisher Scientific
Catalogue number
23250 / 23252
N6666
Type
Colorimetric
Fluorometric
Working range
125 - 2000 µg/mL
10 ng/mL - 10 µg/mL
LYSE
Yes
Yes (dilute 1:100 (v/v) with Assay reagent incl. in the kit)
LYSE-BCT
Yes
Yes (dilute 1:100 (v/v) with Assay reagent incl. in the kit)
LYSE-NHS
Yes
Not compatible
SP3-LYSE 2-fold diluted 1:1 (v/v) with RESUSPEND incl. in the kit
Yes (dilute 1:4 (v/v) with Reconstitution buffer incl. in the kit)
Not compatible
1.8. Which peptides quantification methods are compatible with the PreOmics' kits?
To determine peptide quantity in the sample after using the PreOmics kit family, we recommend using the Pierce™ Quantitative Fluorometric, Pierce™ Quantitative Colorimetric Peptide Assay (Thermo Fisher Scientific). Below is the list of compatible PreOmics buffers and the peptide quantification assay with recommended dilution. Please note that peptide quantification in diluted ELUTE should only be used as a rough estimation. We recommended performing peptide quantification in the LC-LOAD buffer.
Peptide quantification assays and compatible PreOmics buffers
Peptide quantification assays and compatible PreOmics buffers
Assay
Pierce™ Quantitative Fluorometric Peptide Assay
Pierce™ Quantitative Colorimetric Peptide Assay
Manufacturer
Thermo Fisher Scientific
Thermo Fisher Scientific
Catalogue number
23290
23275
Type
Fluorometric
Colorimetric
Working range
5 - 1000 µg/mL
25 - 1000 µg/mL
LC-LOAD
Yes
Yes
ELUTE*
Yes (dilute 1:2 (v/v ) with Water)
Yes (dilute 1:2 (v/v ) with Water)
FRACTION-1**
Yes (dilute 1:2 (v/v ) with Water)
Yes (dilute 1:2 (v/v ) with Water)
FRACTION-2**
Yes (dilute 1:2 (v/v ) with Water)
Yes (dilute 1:2 (v/v ) with Water)
FRACTION-3**
Yes (dilute 1:2 (v/v ) with Water)
Yes (dilute 1:2 (v/v ) with Water)
* Peptide quantification in diluted ELUTE should only be used as a rough estimation. We recommend doing the peptide quantification in LC-LOAD.
** For Fractionation workflow, we recommend Pierce colorimetric assay.
** For Fractionation workflow, we recommend Pierce colorimetric assay.
1.9. What is the maximum volume I can load on the PreOmics’ CARTRIDGES?
The maximum volume is 250 µL.
1.10. Should I adjust the volumes of the buffers with protein starting amounts?
We recommend using the buffer volumes indicated in the protocol. You can adjust the volume of LYSE and DIGEST to your starting amounts. For less than 20 µg protein starting material use the following volumes: 10 µL LYSE; 10 µL DIGEST; keep the other buffers as indicated in the protocol. Accordingly, you may adjust the volumes for chemical labeling and quenching as recommended by the label manufacturer.
1.11. Which lysis temperature shall I use?
Perform the lysis/denaturation step at 95°C. Use lower temperatures only for temperature-sensitive samples such as immunoprecipitations. PreOmics have tested lower temperatures down to 60°C and do not see any differences in parameters assessed (e.g., IDs, alkylation rates).
In case you are working with your own urea-containing lysis buffer, please see FAQ 1.1 for further information.
In case you are working with your own urea-containing lysis buffer, please see FAQ 1.1 for further information.
1.12. Can I use other enzymes for the protein digestion?
The PreOmics’ kits come with a lyophilized enzyme mix consisting of LysC and trypsin, we do not supply other enzyme combinations.
1.13. How long shall I digest my samples?
The digestion depends on your sample type and input material (see table below). Please note our recommendations to lower the enzyme amount when working with low input samples (<20 µg protein starting material, see 1.10.)
Sample type
Digestion time
Precipitation proteins
1-3 hrs
Cell lines
1-3 hrs
Biological fluids
1-3 hrs
Tissues (mammalian, plants)
3 hrs
Although not recommended, you can also digest your samples overnight (~18 hours). While this will reduce the missed cleavage rate even further, it will come at the cost of higher unspecific cleavages and much longer processing time of the overall workflow.
1.14. Are the PreOmics’ workflows compatible with enrichment of phosphorylation sites?
While our protocols are in general compatible with IP samples (modified protocols can be found to download on our website), the required input amounts for global phosphorylation enrichment experiments (~500 µg) usually exceed the peptide binding capacity of our CARTRIDGES.
1.15. How can I automate my sample processing?
The PreOmics' PreON is a dedicated sample preparation platform suitable for 12 or 16 samples, see here or click on the "PreON platform" tab for more information. For automation of up to 96 samples per run, we offer the APP96 system, see here and section 6 for more information. Moreover, PreOmics is working with liquid handling systems providers, and application notes are available here. We are also happy to provide technical assistance to transfer the PreOmics methodology onto your existing platform if appropriate.
1.16. Are the PreOmics kits compatible with absolute quantification?
All of the PreOmicskits are compatible with absolute quantification strategies. For absolute quantification employing isotopically-labeled protein standards, please add the respective absolute standard (e.g. DIGESTIF, PSAQ, SILAC-PrEST) together with your sample to the appropriate LYSE buffer ensuring the total protein does not exceed 100 µg and proceed with the protocol accordingly.
For absolute quantification employing isotopically-labeled peptide standards with the PreOmics' kits, please introduce the respective standard (e.g. AQUA, QconCAT) before adding the STOP buffer.
For absolute quantification employing isotopically-labeled peptide standards with the PreOmics' kits, please introduce the respective standard (e.g. AQUA, QconCAT) before adding the STOP buffer.
1.17. How do I assess the peptide recovery rate after the whole sample processing workflow?
The best way to assess peptide recovery rates is by employing absolute quantification strategies (see 1.8.).
1.18. What kind of quality control does PreOmics provide for the kits?
Production of PreOmics’ kits is ISO 9001:2015 certified. Quality control measures include incoming goods control, polymer leaching tests for plasticware, LC/MS-based quality control for parameters including alkylation rate or missed cleavages on a standardized sample. Upon request, PreOmics can provide a certificate of analysis (CoA) to our clients.
1.19. What are the differences between the 4x, 8x, 12x, 96x and 192x PreOmics’ kits?
Our kits are provided in pack sizes according to the number of samples that can be processed:
iST kits
◦ 4 reactions: test kits for first time users
◦ 8 reactions: small kits for initial experiments
◦ 96 reactions: medium kits with flexibility to process either up to 96 samples at once or multiple smaller batches, as all buffers and enzymes are supplied in 2 mL tubes
◦ 192 reactions: large kit suitable for liquid handling platforms, supplied in 20 mL vials for convenient transfer into liquid handling reservoirs
iST-REG-PSI kits
◦ 192 reactions: large kit suitable for liquid handling platforms, supplied in 20 mL vials for convenient transfer into liquid handling reservoirs, includes 2 fixed well plates for the PURIFY step.
iST-NHS kits
◦ 4 reactions: test kits for first time users
◦ 12 reactions: small kits for initial experiments
◦ 96 reactions: medium kits with flexibility to process either up to 96 samples at once or multiple smaller batches, as all buffers and enzymes are supplied in 2 mL tubes
◦ 192 reactions: large kit suitable for liquid handling platforms, supplied in 20 mL vials for convenient transfer into liquid handling reservoirs
iST-BCT kits
◦ 4 reactions: test kits for first time users
◦ 8 reactions: small kits for initial experiments
◦ 96 reactions: medium kits with flexibility to process either up to 96 samples at once or multiple smaller batches, as all buffers and enzymes are supplied in 2 mL tubes
Phoenix kits
◦ 4 reactions: test kits for first time users
◦ 96 reactions: medium kits with flexibility to process either up to 96 samples at once or multiple smaller batches, as all buffers supplied in 2 mL tubes
iST kits
◦ 4 reactions: test kits for first time users
◦ 8 reactions: small kits for initial experiments
◦ 96 reactions: medium kits with flexibility to process either up to 96 samples at once or multiple smaller batches, as all buffers and enzymes are supplied in 2 mL tubes
◦ 192 reactions: large kit suitable for liquid handling platforms, supplied in 20 mL vials for convenient transfer into liquid handling reservoirs
iST-REG-PSI kits
◦ 192 reactions: large kit suitable for liquid handling platforms, supplied in 20 mL vials for convenient transfer into liquid handling reservoirs, includes 2 fixed well plates for the PURIFY step.
iST-NHS kits
◦ 4 reactions: test kits for first time users
◦ 12 reactions: small kits for initial experiments
◦ 96 reactions: medium kits with flexibility to process either up to 96 samples at once or multiple smaller batches, as all buffers and enzymes are supplied in 2 mL tubes
◦ 192 reactions: large kit suitable for liquid handling platforms, supplied in 20 mL vials for convenient transfer into liquid handling reservoirs
iST-BCT kits
◦ 4 reactions: test kits for first time users
◦ 8 reactions: small kits for initial experiments
◦ 96 reactions: medium kits with flexibility to process either up to 96 samples at once or multiple smaller batches, as all buffers and enzymes are supplied in 2 mL tubes
Phoenix kits
◦ 4 reactions: test kits for first time users
◦ 96 reactions: medium kits with flexibility to process either up to 96 samples at once or multiple smaller batches, as all buffers supplied in 2 mL tubes
1.20. Are the PreOmics' kits compatible with peptide fractionation?
Yes, all our kits are compatible with downstream peptide fractionation workflows. PreOmics has a dedicated iST-Fractionation Add-on kit or alternatively after peptides are dried completely, resuspend the peptides either in LC-LOAD or a resuspension buffer of your choice as input buffer for further peptide fractionation methods.
1.21. When should I use the iST-BCT kit?
The iST-BCT kit has been designed specifically for use with biological fluids plasma and serum samples and for analysis of monoclonal antibodies, therapeutic proteins and host cell proteins (HCPs). The LYSE-BCT provides enhanced reduction and alkylation efficiency and all reagents are optimized to minimize artificial modifications such as oxidation or deamidation.
1.22. What is the difference between iST and iST-BCT kits?
The LYSE-BCT and RESUSPEND-BCT reagent composition has been modified with reduction agent, detergent and pH being optimised to give improved performance. The iST-BCT CARTRIDGE is NOT suitable for sonication and is identical to the PHOENIX CARTRIDGE.
1.23. How can I minimize artificial modifications when using iST-BCT?
For even lower oxidation and deamidation rates and increased alkylation rate, reduce the temperature to 80°C and incubate for 10-20 minutes.
1.24. Do you have to process the samples on the CARTRIDGES provided in the PreOmics’ kits?
You can perform the LYSE and DIGEST steps in an appropriate plate or microcentrifuge tube until the addition of the STOP reagent. The total volume should then be transferred to the CARTRIDGE for the purify steps.
1.25. Are nucleases like benzonase compatible with PreOmics' kits?
Yes, nucleases like benzonase are compatible with all PreOmics' LYSE-buffers, including iST LYSE, LYSE-BCT, LYSE-NHS, and SP3-LYSE. They can be used for various sample types, such as mammalian and bacteria cells, yeast, and tissues. Add the enzyme to the LYSE buffer used for the lysis step.
NOTE: Five units of benzonase per 100 µg protein input is sufficient for efficient DNA cleavage. For lysis of larger protein amounts, adjust the enzyme amount accordingly. Please note that benzonase is incompatible with lysis buffers containing EDTA.
NOTE: Five units of benzonase per 100 µg protein input is sufficient for efficient DNA cleavage. For lysis of larger protein amounts, adjust the enzyme amount accordingly. Please note that benzonase is incompatible with lysis buffers containing EDTA.
1.26. What do I need to consider for the data analysis of my protein sample preparation experiment?
Please consider the following as fixed modifications in your database search:
Product
Modification
Description
Composition
Specificity
Mass
iST/iST-BCT/ENRICH
Alkylation
Carbamidomethyl on cysteine
C2H3NO
[C]
+57.02 Da
iST-NHS
Alkylation
Specific cysteine modification
C6H11NO
[C]
+113.08 Da
1.27. Where do I find the protocol for my kit?
All protocols can be found at www.preomics.com/resources, including general kit protocols and sample-specific protocols. Please contact us at info@preomics.com if you have any questions about specific protocols or your specific sample.
2. Sample type recommendations
2.1. How much raw material do I need for the PreOmics' kits?
Protein content varies considerably across distinct biological input material. Different cell lines, strains, tissues, tissue regions, biological fluids and the sample storage conditions can affect the protein concentration. We recommend carrying out a protein concentration assay of the sample after the lysis step (see 1.7). A short overview of raw material amounts is given in the table below.
We have a database with processing recommendations for >100 species and sample matrices. Please contact us for further details on your specific sample of interest.
We have a database with processing recommendations for >100 species and sample matrices. Please contact us for further details on your specific sample of interest.
Material
Starting amount
Protein amount
Mammalian Cell Line (e.g. HeLa)
6E5 cells
100 µg
Yeast (S.cerevisae)
OD600 = 0.6 (1mL of culture)
100 µg
Bacteria (E.coli)
OD600 = 0.5 (1mL of culture)
100 µg
Immunoprecipitation
1 mL slurry
10 - 400 µg
Blood / Serum / Plasma (H.sapiens)
2 µL
100 µg
Mammalian Tissue
1 mm3
100 µg
Plant Tissue (A.thaliana): shoot/root wet weight
50 mg / 100 mg
100 µg / 100 µg
2.2. How do I process non-depleted plasma/serum samples?
Non-depleted human plasma and serum samples have a high protein concentration, commonly around 50 µg/µL. For processing of non-depleted plasma/serum for non-labeled the iST-BCT kit should be used, for labeled workflows use the iST-NHS kit. Mix 2 µL plasma/serum with 50 µL LYSE-BCT / LYSE-NHS buffer and continue with the regular iST-BCT / iST-NHS protocol.
2.3. How do I process depleted plasma/serum samples?
Depending on the depletion process used, the resulting depleted plasma/serum sample may contain high concentrations of salts and/or have a large volume, making the samples incompatible for direct processing with PreOmics’ kits. We suggest performing protein precipitation (see 1.3) and using the resulting pellet as input material for the iST-BCT/iST-NHS protocol.
2.4. How do I process CSF samples?
CSF samples have a wide reported concentration range, therefore PreOmics suggest performing protein quantification assay to determine the concentration prior to processing (see 1.7). For highly concentrated CSF samples, follow the recommendations for non-depleted plasma/serum (see 2.2) For diluted CSF samples (volume larger than 25 µL), perform protein precipitation (see 1.3) and continue with the regular PreOmics protocol. Both iST and iST-BCT are suitable for unlabeled workflows, iST-NHS should be used for labeled workflows.
2.5. How do I process urine samples?
Protein concentrations in urine samples vary substantially, we recommend either concentrating or precipitating the protein (see 1.3) before processing with the PreOmics kits. As a rule of thumb, 10-100mL human urine corresponds to ~100 µg protein, which needs to be concentrated down to 100 µL. Specifically for urine preparations, PreOmics provides an extra WASH0 buffer to effectively remove bilirubin contamination. Please inquire or refer to our website for an adapted protocol.
2.6. How do I process saliva samples?
Protein concentrations in saliva samples vary widely, a rough estimate is about 10 µg/µL. Either collect ~10 µL saliva by spitting into a microcentrifuge tube or perform mouth swab and place the swap in 50-100 µL LYSE LYSE-NHS buffer to fully cover the swap. Keep all other buffer volumes as indicated in the regular protocols.
2.7. How do I process adherent cell culture samples?
If extracellular matrix or transmembrane proteins are not of interest, you can use cell culture-grade trypsin to detach the adherent cells, wash them once with PBS and then store the cell pellet fraction as input for our regular protocols. Alternatively, if extracellular matrix or transmembrane are of interest and affected by partial trypsin digestion, add 50-100 µL of our LYSE/LYSE-NHS buffer directly to the cells and scrape them off. Collect the scraped material and heat to 95°C for 10 min before continuing with theregular iST/iST-NHS protocol.
2.8. How do I process mammalian tissue samples?
Mammalian tissue samples are more difficult to lyse and require some kind of mechanical force disruption in the presence of our PreOmics’ LYSE buffers (). PreOmics’ BeatBox instrument efficiently and reproducibly homogenizes tissues and lyses cells from various sources. For more information, see https://www.preomics.com/beatbox.
2.9. How do I process FFPE samples?
FFPE samples can be easily processed by combining homogenization on the BeatBox with the iST technology. This workflow does not require a separate deparaffinization step with xylene. More details and a dedicated protocol can be found at https://www.preomics.com/applications/ffpe. FAQs related to this protocol are collected in section 12.
2.10. How do I process plant tissue samples?
Plant tissues can be processed with our iST and iST-NHS kits but require cryogenic grinding or other means of mechanical force disruption initially. Specifically for plant preparations, PreOmics provides an extra WASH0 buffer to effectively remove secondary metabolite contamination, the protocol can be found here.
2.11. How do I process immunoprecipitation samples?
Enrichment of proteins via IP or co-IP strategies is compatible with our iST and iST-NHS technologies. Depending on the type of bead material used, the sample transfer to our cartridges happens either directly after the IP (transfer of proteins; magnetic beads) or after the digestion (transfer of peptides; agarose beads). For further information, please have a look at our two workflow recommendations available on the protocol tab here).
2.12. How do I process bacteria/yeast/algae/diatoms?
Model organisms can be entirely processed with our iST or iST-NHS kits. We recommend mechanical force disruption in the presence of our lysis buffers to effectively disrupt tissues/lyse cells. For processing of algae and diatoms, our WASH0 buffer removes secondary metabolites (provided upon request).
3. iST-NHS for chemical labeling (iTRAQTM, TMTTM)
3.1. What is the difference between the iST and the iST-NHS kits?
The lysis buffer in the iST-NHS kit, called LYSE-NHS, does not contain primary amines and, therefore, does not interfere with chemical labeling. The LYSE-NHS contains a distinct alkylation agent. Please consider the following as fixed modification in your database search:
Specific cysteine modification (C6H11NO), specificity [C], mass shift +113.084 Da
Specific cysteine modification (C6H11NO), specificity [C], mass shift +113.084 Da
3.2. How can I improve the labeling efficiency when performing chemical labeling in combination with the iST-NHS kits?
Chemical labeling experiments require very high peptide labeling efficiencies (>98%) for proper quantification. When struggling with lower labeling efficiencies, please see the following points:
- Make sure that the sample input material (e.g. cell pellet) is not contaminated with residual buffersor cell culture media containing primary amines that interfere with chemical labeling.
- Use fresh labeling reagents to achieve the highest labeling efficiency. Resuspended labeling agent,which is not used throughout the experiment, should not be stored longer than two weeks at -20°C. Resuspended labeling reagent will hydrolyze over time leading to lower labeling efficiency.
- Use TMT at a label-to-peptide ratio of 4:1, i.e., 400 µg of TMT label per 100 µg of peptides. Higher ratios will slightly increase the labeling efficiency but commonly reduce peptide identification rates
- Use an acetonitrile (ACN) concentration of at least 30% during the labeling reaction, i.e., 50 µL LYSE+ 50 µL DIGEST + 42 µL of labeling reagent in dry ACN. Lower amounts of ACN will quickly hydrolyze the labeling agent. If you have resuspended the labeling reagent in a smaller ACN volume, add some dry ACN to the solution in order to achieve a final concentration of 30% ACN.
Take the volume of the cell pellet and residual buffer/cell culture media into account for the calculation of the final ACN concentration.
3.3. When should I mix channels after the chemical labeling?
There are two options for channel mixing:
A. Perform workflow until addition of STOP buffer, then mix channels accordingly. The pooled sample can be equally split on different CARTRIDGES (binding capacity of 100 µg per CARTRIDGE).
B. Perform workflow until the end of the vacuum concentrator step. Resuspend individual channels in LC-LOAD and then pool channels accordingly. We advise measuring the peptide concentration of each channel for optimal channel pooling results.
A. Perform workflow until addition of STOP buffer, then mix channels accordingly. The pooled sample can be equally split on different CARTRIDGES (binding capacity of 100 µg per CARTRIDGE).
B. Perform workflow until the end of the vacuum concentrator step. Resuspend individual channels in LC-LOAD and then pool channels accordingly. We advise measuring the peptide concentration of each channel for optimal channel pooling results.
3.4. Should I perform the chemical labeling step on the cartridge or in the tube?
Perform the labeling in an 1.5 mL microreaction tube and only to transfer the labeled peptides to our CARTRIDGES for the final peptide cleanup.
4. iST-PSI for positive pressure processing
4.1. What is the difference between the iST and iST-REG-PSI HT 192x kits?
The iST-PSI kit contains two fixed well clean-up plates for the purification step on a positive pressure unit. The iST kit clean-up kit has an “array” plate format allowing the individual cartridges to be removed and processed in microcentrifuge tubes using adapters (not included but available for order separately).
4.2. My automation platform has some dead volumes, will there be enough buffers?
Additional buffers for the clean-up steps can be ordered separately if needed.
P.O.00109 iST-REG-PSI buffer add-on contains 2 x 100 mL of WASH1, WASH2 and ELUTE.
P.O.00109 iST-REG-PSI buffer add-on contains 2 x 100 mL of WASH1, WASH2 and ELUTE.
4.3. How can I process the iST-REG-PSI plates?
The iST-REG-PSI plates have been designed to work optimally when used with positive pressure, however they can also be processed by centrifugation.
5. ENRICH technology for plasma and serum sample preparation
5.1. What is the difference between ENRICHplus, ENRICH-iST and iST-BCT kits?
The iST-BCT kit is used to prepare various biological fluid samples for proteomic analysis, such as plasma, serum, and CSF. The ENRICH-iST kit allows deeper insights into the plasma, serum, and CSF proteome. ENRICH-iST combines a bead-based upstream enrichment step with iST-BCT sample preparation. ENRICHplus combines bead-based upstream enrichment with iST clean-up. ENRICHplus is optimized for human EDTA plasma samples by using tailor-made buffers. Compared to ENRICH-iST, a significantly deeper proteome coverage can be achieved with ENRICHplus. Other anticoagulants and biofluids may not work with ENRICHplus.
ENRICH technology is founded on the bead-based enrichment of low-abundant proteins by breaking the dynamic range of plasma/serum proteome. ENRICH-iST combines a bead-based upstream enrichment step with iST-BCT sample preparation.
ENRICH technology is founded on the bead-based enrichment of low-abundant proteins by breaking the dynamic range of plasma/serum proteome. ENRICH-iST combines a bead-based upstream enrichment step with iST-BCT sample preparation.
5.2. When should I use ENRICH-iST?
The ENRICH-iST kit has been specifically designed for plasma and serum sample preparation.
The ENRICH-iST kit should be used when your target protein is low abundant, and its detection— if preparing the sample with iST-BCT—could be challenging.
The ENRICH-iST kit should be used when your target protein is low abundant, and its detection— if preparing the sample with iST-BCT—could be challenging.
5.3. What is the difference between ENRICH-iST 96x and ENRICH-iST 96x HT?
ENRICH-iST 96x:
It has the flexibility to process batches from 1 to 96 samples. The DIGEST solution and buffers of the included iST-BCT are supplied in 2 mL tubes.
ENRICH-iST 96x HT:
It is used for high-throughput analysis to process 48 or 96 samples at once. The DIGEST solution is supplied in one vial, and all buffers are provided in 30 mL vials.
It has the flexibility to process batches from 1 to 96 samples. The DIGEST solution and buffers of the included iST-BCT are supplied in 2 mL tubes.
ENRICH-iST 96x HT:
It is used for high-throughput analysis to process 48 or 96 samples at once. The DIGEST solution is supplied in one vial, and all buffers are provided in 30 mL vials.
5.4. Can I use different anticoagulants common in blood collection with ENRICH-iST?
Yes, ENRICH-iST is compatible with different anticoagulants. We recommend K2 EDTA or K3 EDTA as anticoagulants and blood harvest at RT; plasma should be prepared within 2 hours of blood collection by centrifugation at 2000 g for 10 min.
5.5. Which biological fluids can be used with ENRICH-iST?
Plasma and serum samples derived from humans and other mammalian species (i.e., mice, rats, pigs, and dogs) as well as cerebrospinal fluid (CSF) can be used. Additional sample types, such as, cell culture media have also been tested. Please contact info@preomics.com for further information.
5.6. What is the minimum and maximum input volume for plasma and serum sample preparation with ENRICH-iST?
The recommended plasma or serum input volume is 20 µL. The minimum sample input volume is 10 µL, which might lead to a decrease in the protein identification rate. The maximum sample input volume is 40 µL; however, no dramatic increase in protein identification is observed by increasing the input volume.
5.7. What is the peptide amount obtained from 20 µL of plasma?
With ENRICH-iST, approximately 10 µg peptide can be obtained from 20 µL of plasma/serum.
5.8. Can I automate the ENRICH-iST workflow?
Yes, the ENRICH-iST workflow can be automated on liquid handling systems compatible with magnetic bead handling and can be coupled with a positive pressure unit. We automated ENRICH-iST workflow on Tecan Fluent® Automation Workstation with Resolvex® A200. A corresponding poster is available here (A standardized, high-throughput platform for automated, rapid, and extensive plasma proteome characterization). We also partially automated the ENRICH-iST protocol using the Opentrons OT-2 system. Scripts and quick start guides can be shared upon request. For further information, please contact us at info@preomics.com.
5.9. Can the ENRICH step also be done at RT instead of 30°C?
The protein binding at 30°C is set for the most robust performance of ENRICH-iST. We do not recommend protein binding at RT as the temperature can vary from lab to lab or daily. During ENRICH-iST validation, the binding step was tested from 4 to 37°C without significant changes in the number of proteins identified, but it can affect the kit robustness.
5.10. Can I wash the required amount of beads in batches and distribute them to the samples afterward?
The performance study did not cover washing beads in batches and distributing them afterward, but altered results are not to be expected.
5.11. What is the recommended LC-MS peptide loading amount after performing ENRICH-iST?
This will vary depending on the LC-MS hardware used. We generally load 300 ng of peptides on an EASY-nLC™ 1200 system (Thermo Fisher Scientific) coupled to a timsTOF Pro/HT (Bruker); peptides are separated on a self-packed 30 cm column using a 25 min gradient and acquired using data-independent acquisition.
5.12. How does the ENRICH-iST technology work?
ENRICH-iST technology is based on the physicochemical binding of proteins to the beads in a specifically designed buffer. Further discloser of the ENRICH-iST methodology cannot be shared due to proprietary information.
5.13. Can ENRICH-iST be used with plasma/serum from species other than human?
Yes, ENRICH-iST can be used across multiple species, such as humans, mice, rats, etc.
5.14. Which sample pre-treatments are compatible with ENRICH-iST workflow?
Pre-treatments that induce conformation or structural changes in proteins affect the efficiency of protein enrichment on EN-beads. Agents or workflows/treatments that denature proteins, such as heat exposure, protein precipitation, or the use of denaturing detergents are incompatible with ENRICH-iST.
5.15. What is the size of the ENRICHplus kit?
ENRICHplus is offered as a 12x test size format and a 96x HT kit solution.
5.16. Can I use the 96x HT kit also for smaller sample sets?
ENRICHplus 96x HT can be split into 4x 24 reactions.
5.17. What is the minimum and maximum input volume for human EDTA plasma with ENRICHplus?
The recommended plasma input volume is 50 µL. The minimum sample input volume is 20 µL, which might lead to a decrease in the protein identification rate. The maximum recommended sample input volume is 80 µL; however, no dramatic increase in protein identification is observed by increasing the input volume.
5.18. What is the peptide amount obtained from 50 µL of human EDTA plasma?
With ENRICHplus, approximately 3-4 µg peptide can be obtained from 50 µL of plasma.
5.19. Can I automate the ENRICHplus workflow?
ENRICHplus workflow can be automated on liquid handling systems compatible with magnetic bead handling and can be coupled with a positive pressure unit. We combined ENRICHplus workflow on KingFisherTM Flex with eighter plate centrifugation for the clean-up part or with Tecan Fluent® Automation Workstation Resolvex® A200. ENRICHplus can be executed manually, semi-automated, or fully automated. Scripts and quick start guides can be shared upon request. For further information, please contact us at info@preomics.com.
6. Peptide Cleanup
6.1. Which sample types require additional wash steps?
Most peptide samples can be cleaned up with the washing buffers in the PreOmics’ kits. However, for certain samples such as urine or plant tissues, our regular iST or iST-NHS protocols can be extended with one additional washing buffer called “WASH0” to remove metabolites. When preparing peptide samples with high levels of contamination, e.g. sugars, fat, polymers or high concentrations of detergents, we recommend to use our PHOENIX peptide cleanup kit instead which contains an additional wash step with “WASHX”.
6.2. What are the differences between the PreOmics washing buffers?
The Phoenix peptide cleanup kit is able to remove detergents, sugars, lipids, salts and various polymers. For more details see our Phoenix Application Note.
Washing buffer
Organic
Acidic
Basic
Volatile
Sample types
WASH0
Yes
Yes
-
Yes
urine, plants, FFPE
WASHX
Yes
Yes
-
Yes
lipids, polymers, detergents
WASH1
Yes
Yes
-
Yes
hydrophobic contaminations
WASH2
-
Yes
-
Yes
hydrophilic contaminations
6.3. How do I elute my samples from the 96-well CARTRIDGE adapter plates?
You can either stack the 96-well CARTRIDGE adapter plate on top of the provided collection plate,or you can stack it on top of standardized autosampler vials. To avoid damage to the collection plate wells during the centrifugation step, use a suitable plate carrier together with the swing-bucket rotor (e.g. PCR plate adapter Eppendorf #5825711009 for Eppendorf plate rotors).
6.4. How long do I need to concentrate my samples in the vacuum concentrator?
Concentrate at 45°C until the sample is dry and no residual ELUTE buffer is left. This usually takes about 30 min. Depending on your sample, peptide ions might accumulate at the top layer and thus interfere with efficient evaporation. To overcome this, tap the sample briefly to mix the eluate and then continue to concentrate in the vacuum concentrator.
6.5. Can I concentrate PreOmics’ eluates together with samples eluted from C18 columns in the same vacuum concentrator?
Since our ELUTE buffer has a basic pH and C18 eluates have an acidic pH, do not place them in the same vacuum concentrator as this can damage the instrument.
6.6. Which peptide quantification methods can I use after processing my samples?
Peptide quantification should be done in our LC-LOAD buffer and not in the ELUTE buffer. We recommend quantitative colorimetric peptide assays.
6.7. How do I resuspend dried peptides in the 96-well plate?
After the vacuum concentrator step, you can add our LC-LOAD buffer to each well and shake in a horizontal plate shaker (500 rpm, 5 min).
6.8. How should I store resuspended peptide samples after processing them?
Storage of peptides should not exceed two weeks at -20°C. For long-term storage, keep them at -80°C.
7. Phoenix kit for peptide cleanup
7.1. Are the CARTRIDGES from all the PreOmics' kits the same?
CARTRIDGES in the iST and iST-NHS kits are the same. CARTRIDGES in the iST-BCT and PHOENIX kits are of different composition and have a slightly lower affinity for hydrophobic species.
7.2. How do I load my peptide samples on the PHOENIX CARTRIDGES?
It is essential to acidify the peptides, otherwise your sample will not bind to the CARTRIDGE. To do so, mix your peptides 1:1 with the provided STOP buffer and load everything on the CARTRIDGE.
Spin at 3,800 rcf for 1-3 min to load the sample completely.
Note: pH of STOP itself cannot be determined by pH measurement device or pH paper due to a high concentration of organic solvent. After adding 100 µL STOP to the sample, pH can be measured by pH paper.
Spin at 3,800 rcf for 1-3 min to load the sample completely.
Note: pH of STOP itself cannot be determined by pH measurement device or pH paper due to a high concentration of organic solvent. After adding 100 µL STOP to the sample, pH can be measured by pH paper.
8. iST-Fractionation Add-on kit for peptides fractionation
8.1. What is in the iST-Fractionation Add-on kit?
The iST-Fractionation Add-on kit contains three buffers, please note it does NOT contain the CARTRIDGE or LC-LOAD reagent, therefore this kit should ALWAYS be used in conjunction with one of the iST family of kits.
8.2. Is the iST-Fractionation Add-on compatible with the iST-BCT kit and Phoenix clean-up kit?
Yes, the iST-Fractionation Add-on kit is compatible with all of the PreOmics iST family of kits.
8.3. How much LC-LOAD should I use for each fraction?
Add LC-LOAD to COLLECTION tubes 1-3, typically we suggest that you aim for 1 g/L concentration remembering that the starting protein concentration will have been fractionated into 3 (e.g. 90 µLto 90 µg protein starting material equates to 30 µL per tube).
8.4. Are the PreOmics' kits compatible with other peptide fractionation protocols?
Yes, all our kits are compatible with downstream peptide fractionation workflows. After peptides are dried completely, resuspend the peptides either in LC-LOAD or a resuspension buffer of your choice as input buffer for further peptide fractionation methods.
9. SP3-iST Add-on kit for SP3-based workflows
9.1. What is in the SP3-iST Add-on kit?
The SP3-iST Add-on kit contains SP3 beads and reagents for bead preparation, non-biased protein binding, lysis and washing. The kit must be used in conjunction with one of the iST, iST-NHS or iST-BCT kits.
9.2. Which protein extraction buffers are compatible with the SP3-iST Add-on kit?
Reagent name or type
Tested concentration range
Considerations for performing SP3 in the described conditions
Detergents
0-20%
Detergents tested in the listed concentration range include SDS, Triton X-100, NP-40, Tween 20, deoxycholate, CHAPS, and RapiGest. We recommend keeping the total detergent concentration in the range of 0–10% (wt/vol or vol/vol depending on the detergents used)
Chaotropes
0-8 M
Chaotropes tested in the listed concentration range include urea (up to 8 M), guanidinium hydrochloride, and isothiocyanate (up to4 M). High concentrations of chaotropes in the presence of the solvent used for binding can disrupt the interactions between SP3 beads and the proteins. Therefore, we recommend testing SP3 with your desired lysis solution formulation before further application
Salts
0-1 M
A wide range of salts have been tested, and we recommend keeping the final salt concentration in the lysate <1 M when using solvents for binding. High salt concentrations in the presence of binding solvent can disrupt the ability of proteins to efficiently localize on the SP3 bead surface. The critical concentration is going to depend on the identity and properties of the salt in question
Solvents
0-50%
Solvents tested in the listed concentration range include acetonitrile, acetone, isopropanol, ethanol, trifluoroethanol, and xylene. We recommend keeping the final solvent concentration in the lysate before binding <25% (vol/vol). If high solvent concentrations are present, the amount of ethanol added during SP3 can be scaled to achieve the desired final solvent concentration (e.g., 50% (vol/vol) final)
Hughes, C.S., Moggridge, S., Müller, T. et al. Single-pot, solid-phase-enhanced sample preparation for proteomics experiments. Nat Protoc 14, 68–85 (2019). https://doi.org/10.1038/s41596-018-0082-x
For urea-containing buffers: To reduce the risk of carbamylation of proteins, it is recommended not to heat samples above 37 °C in urea-containing buffers. Please contact us via info@preomics.com for recommendations on how to modify the protocol.
For urea-containing buffers: To reduce the risk of carbamylation of proteins, it is recommended not to heat samples above 37 °C in urea-containing buffers. Please contact us via info@preomics.com for recommendations on how to modify the protocol.
9.3. How do I use my own extraction buffer with the SP3-iST Add-on kit?
Prepare your samples in your own extraction buffer (maximum volume 50 µL) and use the SP3 protocol as given, ensuring in step 2.1 that you add 50 µL of SP3 LYSE and make up to 100 µL with RESUSPEND if needed.
9.4. Do I have to use my own extraction buffer or can I just use SP3 LYSE?
You do not have to use a combination of buffers as the protocol will work successfully using SP3 LYSE alone. Add 50 µl of SP3 LYSE to your sample and make up to 100 µl with RESUSPEND.
9.5. Does the SP3 LYSE contain detergents, reducing and alkylation agents?
The SP3 LYSE buffer contains detergent and reduction agents needed. The alkylation reagent is added in the appropriate kit LYSE reagent.
9.6. Is the SP3 LYSE buffer compatible with protein determination assays?
The SP3 LYSE is not compatible with the BCA assay, we recommend starting with your usual extraction buffer and protein assay, if you do not have a preferred assay then we suggest the Detergent Compatible Bradford Assay from ThermoFisher https://www.thermofisher.com/de/en/home/life-science/protein-biology/protein-assays-analysis/protein-assays/bradford-assays.html
9.7. How do I know what SP3 bead volume to use?
The bead volume is calculated from the number of samples being prepared and the protein concentration of those samples.
For each sample starting protein concentration see the SP3 volume below:
For each sample starting protein concentration see the SP3 volume below:
Protein input amount
Required volume of SP3 BEADS
1 – 10 µg
10 µL
11 – 50 µg
50 µL
51 – 100 µg
100 µL
E.g. for 3 samples containing 50 µg protein, transfer 3x 50 µL of SP3 BEADS
9.8. Can we digest in solution rather than on-bead?
Yes, an aqueous elution step can be performed and may be useful when using the iST-BCT kit. Adjust the pH of sample to pH 8-9 with NaOH solution (added volume should not exceed 10 µL) and shake sample (RT; 1400 rpm; 5 min), then place the sample on the magnetic separator and remove the supernatant to a clean tube for the DIGEST step.
IMPORTANT: After the addition of STOP, make sure that the pH of the sample is acidic (pH 3-4).
IMPORTANT: After the addition of STOP, make sure that the pH of the sample is acidic (pH 3-4).
10. PreON® and APP96™ automation platforms
10.1. What are the PreON pre-installation requirements?
We offer a full pre-installation requirement documentation upon request. Briefly, the PreON operates at: 100–240 V AC, 50/60 Hz, 650 VA. PreON dimensions are (WxDxH): 65 cm (25.6 in.) x 62 cm (24.4 in.) x 86 cm (34.0 in.) with a weight of 71.5 kg (157.6 lbs).
10.2. Which sample types can be processed on the PreON?
The PreON can process protein cell and model organism pellets, body fluids and tissue lysates.
10.3. How many samples can I process on the PreON per day?
The PreON sample throughput is 12 label-free samples per run, or 16 samples in conjunction with TMTpro labeling reagents. An optional upgrade is also available, allowing PreON to run up to 18 samples by using TMTpro 18 plex labeling reagents. For iST label-free workflows, up to three runs per working day are feasible, to process in total up to 36 samples. For iST-NHS workflows, up to two runs per day are possible, for a total of 32 samples (36 samples if the TMT18plex upgrade is installed).
10.4. Can the PreON prepare both label-free and chemical labeling samples?
Yes, the PreON can execute workflows for both label-free and chemical labeling samples with our ready-to-go iST, iST-BCT and iST-NHS kits.
10.5. What tubes should be used in the PreON for samples?
PreOmics recommends the use of 1.5 mL Lo-Bind Eppendorf tubes (Catalog No. 0030108442). Tubes that are not the correct dimensions may cause pipetting errors.
10.6. Can I run my own methods on the PreON?
The PreON works seamlessly with our ready-to-go iST, iST-BCT and iST-NHS kits. Other methods or commercial products cannot be used with the PreON.
10.7. Which kind of maintenance does the PreON require?
The PreON requires minimal maintenance such as cleaning the surfaces and emptying the solid and liquid waste containers. More detailed instructions can be found in the PreON software user interface.
10.8. How is the PreON serviced?
Our trusted partner OneService is a leading global provider of technical services for medical, analytical and industrial equipment. Contact us to inquire about further servicing options and pricing.
10.9. When should I select on-board or off-board lysis on the PreON?
PreON on-board lysis is the default setting when selecting the following sample types: Pellet, Liquid concentrated, or Liquid diluted.
PreON on-board lysis consists of the following steps:
- Load PreON workdeck including your samples according to the instructions in the menu.
- LYSE buffer is added to the sample.
- Samples are heated (83 °C, 10 min, shaking) to effectively extract, denature and reduce and alkylate proteins.
- NOTE: If you expect your samples to be viscous due to DNA released from the nuclei, nucleases can be used to reduce the viscosity of the samples, such as Benzonase. For more information and recommendations on the use of nuclease, see FAQ 1.25.
PreON off-board lysis should be selected in the PreON menu when:
- LYSE buffer (50 µL) has already been added to samples manually.
- Working with samples that require a different lysis temperature (PreON lysis temperature: 83 °C).
- Working with samples that require mechanical homogenization/lysis, such as rigid tissue samples.
Differences between on-board and off-board lysis on the PreON:
PreON on-board lysis consists of the following steps:
- Load PreON workdeck including your samples according to the instructions in the menu.
- LYSE buffer is added to the sample.
- Samples are heated (83 °C, 10 min, shaking) to effectively extract, denature and reduce and alkylate proteins.
- NOTE: If you expect your samples to be viscous due to DNA released from the nuclei, nucleases can be used to reduce the viscosity of the samples, such as Benzonase. For more information and recommendations on the use of nuclease, see FAQ 1.25.
PreON off-board lysis should be selected in the PreON menu when:
- LYSE buffer (50 µL) has already been added to samples manually.
- Working with samples that require a different lysis temperature (PreON lysis temperature: 83 °C).
- Working with samples that require mechanical homogenization/lysis, such as rigid tissue samples.
Differences between on-board and off-board lysis on the PreON:
Addition of LYSE buffer
PreON on-board heating (83 °C, 10 min, shaking)
On-board lysis
Automated by PreON
Yes
Off-board lysis
Manually (50 µL LYSE buffer)
Optional
10.10. How long does the PreON on-board lysis take?
For the PreON on-board lysis, the samples are incubated for 10 min at 83 °C with shaking. Heating the shaker to 83 °C and cooling it down to 37 °C for subsequent digestion takes an additionally ~15 min and ~25 min, respectively. The total time for the PreON on-board lysis step is therefore ~50 min.
10.11. How long can the APP96 operating solution (OS) be used?
Based on our use and testing, OS is very stable and can be used for up to 2 weeks. To ensure optimal performance, we recommend cleaning the OS bottle thoroughly each time before refilling it with fresh OS.
10.12. How stable are the reagents in the iST APP96 and iST-BCT APP96 kits?
a. iST reagents (incl. resuspended iST-DIGEST) can be kept on deck if the ‘cooling to 10°C’ is active or be stored in the fridge for up to 5 days.
b. Resuspended iST-BCT DIGEST should be used as soon as possible. Avoid storing on the deck or in the fridge for over 12 hours.
b. Resuspended iST-BCT DIGEST should be used as soon as possible. Avoid storing on the deck or in the fridge for over 12 hours.
10.13. The APP96 uses the same needle for repeated sample preparation steps. Is there any risk of cross-contamination?
The APP96 employs the same needle for multiple sample preparation steps; however, it includes several thorough washing steps for the needle, both inside and outside, after each sample handling step. Cross-contamination between samples has been thoroughly tested, and no contamination was detected.
10.14. Can the SAMPLE PLATE and foil provided with the APP96 kits be reused?
a. Yes, the SAMPLE PLATE can be reused across multiple days. For example, you can use positions 1 to 12 on Day 1 and positions 13 to 30 on Day 2.
b. The plate foil can also be reused. If you prefer not to cut or reuse the foil, additional foils can be ordered here:
b. The plate foil can also be reused. If you prefer not to cut or reuse the foil, additional foils can be ordered here:
10.15. How many samples can be processed with one reagent bottle?
One reagent bottle contains enough reagent to process up to 48 samples. However, processing smaller batches may require additional reagent due to dead volumes. We recommend using each reagent bottle for no more than 6 runs (e.g., 8 samples/run, totaling 48 samples).
10.16. I see some condensation in the Tray Cooler. Can this be reduced?
Yes, the condensation in the Tray Cooler can be reduced. Since the Tray Cooler can cool down to 20°C below the surrounding temperature, higher temperature differences will cause more condensation. To minimize this, place the Tray cooler in a room with air conditioning, low humidity, and avoid direct sunlight.
10.17. Can I perform only a peptide cleanup or skip the LYSE step on the APP96?
a. You can skip the LYSE and DIGEST steps by de-selecting them when editing the method. However, samples must first be processed using iST or iST-BCT.
b. It is also possible to do Digest & Cleanup and perform the lysis step offboard, e.g., if you prefer different lysis conditions.
b. It is also possible to do Digest & Cleanup and perform the lysis step offboard, e.g., if you prefer different lysis conditions.
10.18. I want to delete a method but get an error message “method cannot be deleted”. How should I proceed?
This error occurs when the method is still assigned to a Job. For safety reasons, methods cannot be deleted while in use. To delete a method, first delete all Jobs, and then you will be able to remove it.
10.19. How long does it take for the Tray Cooler to cool down to 10°C?
This takes roughly 1.5 hours, depending on the surrounding temperature. If needed, you can leave the system running for extended periods, such as overnight, to have it ready for use early the next day.
10.20. Can the instrument run unattended overnight?
Yes, you can start a run in the afternoon and collect your eluted peptides the next morning, for example. In this case, please make sure you use the cooled elution position.
10.21. Are samples processed overnight and kept in ELUTE buffer of the APP96 still of the same quality as those processed immediately after the run?
After approximately 15 hours of overnight storage in the ELUTE buffer, protein and peptide identifications remain comparable, and no significant increase in deamidation rates was observed.
10.22. In a 96-sample run, is there any quality loss between samples 1 and 96?
Based on our internal tests, there is no significant quality loss between samples 1 and 96. Differences in protein/peptide identifications or other QC parameters are negligible, ensuring consistent result quality across all 96 samples.
10.23. What are the APP96 processing times?
The automated processing times (h:mm) from lysis to eluted peptides are 3:40, 5:10, and 8:12 for 24, 48, and 96 samples, respectively.
10.24. Can I use a 96-well cell culture plate directly as a sample plate?
We recommend transferring cell pellets to the provided Deepwell SAMPLE PLATE. A dedicated Deepwell plate adapter is fixed on the thermal mixer. A standard 96-well cell culture plate would not accommodate sufficient volume for the iST workflow, would not fit the thermal shaker adapter, and would fall outside the optimized calibration points.
10.25. What is the APP96 POPtipsTM loading capacity?
The protein input range is 1–100 µg.
11. BeatBox instrument
11.1. What is the wet weight of the tissue I can process with the BeatBox?
For the BeatBox Tissue Kit 96x, the recommended wet weight of tissue is from 1 to 5 mg.
For the BeatBox Tissue Kit 24x, the recommended wet weight of tissue is from 5 to 50 mg.
For the BeatBox Tissue Kit 24x, the recommended wet weight of tissue is from 5 to 50 mg.
11.2. What amount of cell samples can be processed (e.g., cell count)?
Cell count is based on the OD read out. Our recommendation for the BeatBox Tissue Kit 96x would be to use:
- yeast cells, 0.6 OD600, 1 mL volume
- bacteria cells, 0.5 OD600, 1 mL volume
- 6e5 mammalian cells
For the BeatBox Tissue Kit 24x, up to 1e7 mammalian cells can be used.
- yeast cells, 0.6 OD600, 1 mL volume
- bacteria cells, 0.5 OD600, 1 mL volume
- 6e5 mammalian cells
For the BeatBox Tissue Kit 24x, up to 1e7 mammalian cells can be used.
11.3. What are the minimum and maximum volumes of the lysis buffer per well/tube?
We recommend a volume range from 50 to 100 µL of LYSE buffer or your own lysis buffer per well in the BeatBox Tissue Kit 96x.
If you want to decrease the buffer volume for very low sample inputs containing 1-20 µg protein, please contact us via info@preomics.com for a modified protocol.
For the BeatBox Tissue Kit 24x, we recommend 300 to 1000 µL. To create optimal conditions, buffer to sample ratios should be adjusted individually. Lower buffer volumes down to 100 µL are possible but recovering the full sample volume may be difficult for foamy or highly concentrated homogenates.
If you want to decrease the buffer volume for very low sample inputs containing 1-20 µg protein, please contact us via info@preomics.com for a modified protocol.
For the BeatBox Tissue Kit 24x, we recommend 300 to 1000 µL. To create optimal conditions, buffer to sample ratios should be adjusted individually. Lower buffer volumes down to 100 µL are possible but recovering the full sample volume may be difficult for foamy or highly concentrated homogenates.
11.4. What type of tissue can I process with the BeatBox?
BeatBox homogenizes various tissue types, from softer tissues, such as the brain, to more fibrous tissues, such as cardiac muscle. Tissue can be collected from different organisms, e.g. human, rat, mouse, plant.
For hard tissues, such as bone, skin, nails, and hair, protein extraction efficiency is relatively low and requires multiple runs with BeatBox. For bone samples, demineralization prior to homogenization is necessary.
For hard tissues, such as bone, skin, nails, and hair, protein extraction efficiency is relatively low and requires multiple runs with BeatBox. For bone samples, demineralization prior to homogenization is necessary.
11.5. What cell types can I lyse using the BeatBox?
All cell types from procaryotic to eucaryotic cells. When yeast is used, it is recommended that prior to homogenization, the cell slurry is heated for 10 min at 95 °C to break the cell walls.
11.6. What is the protein amount in the sample that the BeatBox can process?
For the BeatBox Tissue Kit 96x, we recommend a maximum of 500 µg of total protein and 5 mg of wet weight for tissue samples. The amount of extracted proteins per mg of tissue wet weight will depend on the tissue type. For cell samples, we recommend a maximum of 100 µg of total protein amount. See FAQ 2.1 for approximate starting amounts corresponding to 100 µg protein.
For low protein input amounts of 1-20 µg, please contact us via info@preomics.com for a modified protocol.
For low protein input amounts of 1-20 µg, please contact us via info@preomics.com for a modified protocol.
11.7. What is the recommended length of homogenization?
This will depend on the tissue type and amount; we recommend a minimum of 5 minutes. In general, softer tissues and smaller tissue amounts will be completely homogenized in one run of 10 min with standard settings. Highly fibrous tissues or bigger tissue chunks may need additional or longer runs for complete homogenization.
11.8. Can I process FFPE tissues with the BeatBox?
Yes, BeatBox can homogenize FFPE tissue. For a dedicated protocol and more information, please visit: https://www.preomics.com/applications/ffpe. FAQs related to this protocol are collected in section 12.
11.9. Can I use my own lysis buffers?
Yes, you can use your own lysis buffers. In general, we recommend LYSE buffer from the PreOmics portfolio or RIPA buffer, since we obtained very good results in terms of protein extraction efficiency in our in-house tests. If you are using your own lysis buffer, you can continue with your in-house sample preparation or with PreOmics kits (subject to composition compatibility – for recommendations and limitations, see FAQ 1.1).
NOTE: If you want to freeze surplus homogenate prepared with the BeatBox Tissue Kit 24x, we recommend using RIPA buffer or SP3-LYSE for lysis and subsequent storage. The PreOmics’ iST-LYSE buffer is incompatible with freezing of tissue homogenates/cell lysates in BeatBox TUBES due to increased protein amounts.
NOTE: If you want to freeze surplus homogenate prepared with the BeatBox Tissue Kit 24x, we recommend using RIPA buffer or SP3-LYSE for lysis and subsequent storage. The PreOmics’ iST-LYSE buffer is incompatible with freezing of tissue homogenates/cell lysates in BeatBox TUBES due to increased protein amounts.
11.10. Can I use tissue samples that have been stored at -80 °C?
Yes, you can use samples that have been stored at -80 ºC. Samples should be thawed on ice before adding lysis buffer. We recommend keeping deep-frozen samples on ice before adding lysis buffer. If you want to freeze samples in the 96w PLATE or the TUBES, do not use liquid nitrogen.
NOTE: PreOmics LYSE buffer’s efficiency to lyse, reduce and alkylate sample rapidly decreases when used below room temperature.
NOTE: PreOmics LYSE buffer’s efficiency to lyse, reduce and alkylate sample rapidly decreases when used below room temperature.
11.11. Can I centrifuge the BeatBox 96w PLATE or the BeatBox TUBES with GYUTO BEADS after homogenization?
Yes, this is possible. It is very important to spin down the BeatBox 96w PLATE at 300-500 rcf for 30-60 sec before removing the SILICONE MAT to avoid cross-contamination. If SP3 LYSE or high concentrations of SDS have been used in your own lysis buffer, a longer spin down time is recommended to decrease the foam, before transferring samples to clean tubes/plate.
The same applies to the BeatBox TUBES from the BeatBox Tissue Kit 24x. Here, we recommend a spin down at 1500 rcf for 30-60 sec.
NOTE: Please do not exceed the following max. centrifugation speeds to avoid plate/tube breakage and sample loss:
- BeatBox 96w PLATE with GYUTO BEAD in swing-bucket rotor: 500 rcf
- BeatBox 96w PLATE without GYUTO BEAD in swing-bucket rotor: 2250 rcf
- BeatBox TUBES with GYUTO BEAD: 1500 rcf
- BeatBox TUBES without GYUTO BEAD: 18000 rcf
Use a suitable plate carrier together with the swing-bucket rotor, e.g. PCR plate adapter Eppendorf #5825711009 for Eppendorf plate rotors.
The same applies to the BeatBox TUBES from the BeatBox Tissue Kit 24x. Here, we recommend a spin down at 1500 rcf for 30-60 sec.
NOTE: Please do not exceed the following max. centrifugation speeds to avoid plate/tube breakage and sample loss:
- BeatBox 96w PLATE with GYUTO BEAD in swing-bucket rotor: 500 rcf
- BeatBox 96w PLATE without GYUTO BEAD in swing-bucket rotor: 2250 rcf
- BeatBox TUBES with GYUTO BEAD: 1500 rcf
- BeatBox TUBES without GYUTO BEAD: 18000 rcf
Use a suitable plate carrier together with the swing-bucket rotor, e.g. PCR plate adapter Eppendorf #5825711009 for Eppendorf plate rotors.
11.12. How do you remove the samples after homogenization?
For the BeatBox Tissue Kit 96x, place the BeatBox 96w PLATE onto a GYUTO BEADS COLLECTION RACK after the homogenization process and centrifugation step. This will pull the beads to the side, and you can then transfer the homogenate by pipetting it into a new reaction vessel/plate. For high throughput applications, we recommend using a multi-channel pipet to reduce the processing time.
For the BeatBox Tissue Kit 24x, spin down the BeatBox TUBES (1500 xg, 30-60 sec) after homogenization and transfer the homogenate using a pipet.
For the BeatBox Tissue Kit 24x, spin down the BeatBox TUBES (1500 xg, 30-60 sec) after homogenization and transfer the homogenate using a pipet.
11.13. What’s the size of the GYUTO BEAD?
This is proprietary information that cannot be shared.
11.14. Are proteins binding on the GYUTO BEAD during homogenization?
The GYUTO BEAD is coated with a surface material that has been designed to prevent protein binding.
11.15. Does the BeatBox have a heating/cooling function?
The BeatBox does not have heating or cooling options. The BeatBox operates at room temperature. For workflows that require cooling during homogenization, samples can be temporarily cooled with cooling racks from other suppliers. Please contact us at info@preomics.com for more information.
11.16. Can the BeatBox be operated in a cold room?
Unfortunately, this is not possible. The BeatBox should be operated at an ambient temperature of 18-28 °C. For workflows that require cooling during homogenization, samples can be temporarily cooled with cooling racks from other suppliers. Please contact us at info@preomics.com for more information.
11.17. Is the sample heated during homogenization in the BeatBox?
During homogenization, the BeatBox operates with minimal heat induction. The temperature of the sample will therefore remain stable at room temperature during the homogenization process.
NOTE: Phosphatase inhibitor cocktails can be added into the PreOmics’ LYSE if needed. For recommendations regarding protease inhibitors, please have a look at FAQ 1.4.
NOTE: Phosphatase inhibitor cocktails can be added into the PreOmics’ LYSE if needed. For recommendations regarding protease inhibitors, please have a look at FAQ 1.4.
11.18. Is it possible to boil samples in the BeatBox96w PLATE? If yes, for how long?
Yes, the BeatBox 96w PLATE containing samples can be heated up to 95 ºC. The maximum time is 6 hours. To avoid SILICONE MAT peeling caused by temperature, we recommend to use a ThermoCycler/PCR Cycler with a heated lid exerting pressure from the top. Alternatively, close the 96w PLATE with cap strips (Eppendorf #0030124847).
NOTE: Spin down the BeatBox 96w PLATE before removing SILICONE MAT to prevent cross-contamination. We recommend to centrifuge the plate for 30-60 seconds at amax. speed of 500 xg.
NOTE: Spin down the BeatBox 96w PLATE before removing SILICONE MAT to prevent cross-contamination. We recommend to centrifuge the plate for 30-60 seconds at amax. speed of 500 xg.
11.19. What type of shaking/sonicating is this? Is it closer to shaking like a bead mill?
BeatBox employs a proprietary technology which homogenizes samples differently in comparison to sonication or bead beating. The highly efficient homogenization is based on the movement of a single magnetic particle, called GYUTO BEAD, in an alternating magnetic field inside a reaction vessel and the resulting disruption of tissues and cells within the sample to be homogenized.
In contrast to bead mills, the only moving part of the BeatBox is the GYUTO BEAD in the plate well or tube. This innovative technology minimizes heat induction in the sample, thereby negating the need for sample cooling.
In contrast to bead mills, the only moving part of the BeatBox is the GYUTO BEAD in the plate well or tube. This innovative technology minimizes heat induction in the sample, thereby negating the need for sample cooling.
11.20. What do the “speeds” refer to? What does low, standard, and high setting mean?
The BeatBox settings of low, standard, and high refer to the power of homogenization and the speed or “intensity” of the GYUTO BEAD movement in the well. The precise mechanism underlying this movement is proprietary.
We recommend starting with the standard setting, which is generally sufficient to completely homogenize softer tissues within 10 min. Harder or highly fibrous tissues may need additional or longer runs and/or the high setting for complete homogenization.
We recommend starting with the standard setting, which is generally sufficient to completely homogenize softer tissues within 10 min. Harder or highly fibrous tissues may need additional or longer runs and/or the high setting for complete homogenization.
11.21. Can we use plates that are not the BeatBox 96w PLATE?
The supplied BeatBox PLATE is optimized to work with the GYUTO BEAD. Homogenization efficiency can be affected if the plate used does not have the correct dimensions. The ratios between the GYUTO BEAD:volume:well shape/size is critical to efficient homogenization and although another plate may appear similar, individual dimensions may not be. PreOmics can recommend alternative plates. Please contact us via info@preomics.com However, PreOmics will not support any issues with possible cross-contamination, leaching or loss of samples, when switching to a different plate or when transferring the GYUTO BEAD.
11.22. Can I use 1.5 mL tubes or my own 2 mL tubes instead of the 2 mL tubes provided with the BeatBox Tissue Kit 24x?
The BeatBox is not compatible with 1.5 mL tubes that have a conical shape, e.g., Eppendorf # 0030120086. The ratios between the GYUTO BEAD:volume:well shape/size are critical to efficient homogenization and have been optimized for the 2 mL tube supplied with the PreOmics kit. PreOmics can recommend alternative 2 mL tubes. Please contact us via info@preomics.com. However, PreOmics will not support any issues with possible cross-contamination, leaching or loss of samples, when switching to different tubes or when transferring the GYUTO BEAD.
11.23. Would it be possible to use the BeatBox for genomics, metabolomics, lipidomics and other omics applications?
The BeatBox has been designed primarily for proteomics and each part of the kits is compatible with common lysis buffers used in these workflows.
However, the BeatBox can also be used for other omics applications, such as metabolomics or lipidomics. Depending on the application, the extraction buffer or solvent may vary. The following compositions are generally compatible with the BeatBox kits:
- aqueous buffers with a maximum of 5% SDS
- organic solvents: methanol or isopropanol (up to 80% in water or acetonitrile)
Other organic solvents commonly used in metabolomics, such as chloroform, MTBE, or ethyl acetate are incompatible with the plastic ware of the BeatBox kits. For further information, please contact us at info@preomics.com.
However, the BeatBox can also be used for other omics applications, such as metabolomics or lipidomics. Depending on the application, the extraction buffer or solvent may vary. The following compositions are generally compatible with the BeatBox kits:
- aqueous buffers with a maximum of 5% SDS
- organic solvents: methanol or isopropanol (up to 80% in water or acetonitrile)
Other organic solvents commonly used in metabolomics, such as chloroform, MTBE, or ethyl acetate are incompatible with the plastic ware of the BeatBox kits. For further information, please contact us at info@preomics.com.
11.24. Is it possible to transfer the BeatBox 96w PLATE directly to an automation system, such as the Hamilton, Opentron, or Tecan for the following sample preparation process steps?
Yes, this is possible after the GYUTO BEADS have been removed from the wells. Simultaneous removal of all 96 GYUTO BEADS can be done with help of the BeatBox bead remover. Please contact us for more details via info@preomics.com.
11.25. Which protein quantification method would you recommend using after BeatBox homogenization?
When PreOmics LYSE buffer is used, we recommend using the Pierce™ BCA Protein Assay Kit - Reducing Agent Compatible or the NanoOrange™ Protein Quantitation Kit (Thermo Fisher Scientific), depending on which LYSE is used. Some assays require dilution with distilled water to achieve the best results, please see FAQ 1.7. for more information. If using your own lysis buffers, be aware of any of the maximum concentrations and their compatibility with the assay of choice. Again, dilution may be needed to prevent interference of the readout.
11.26. Protein quantification methods require a clear lysate. What would be your recommendation to achieve this?
For the protein quantification step, mix the tissue homogenate/cell lysate by pipetting, transfer an appropriate amount of mixture to the new reaction vessel and centrifuge at 10 000 xg to pellet the debris. The clear supernatant can be used for protein quantification.
11.27. What is moving in the BeatBox during the homogenization process?
The only thing that moves during the process is the GYUTO BEAD.
11.28. Is polymer released from BeatBox 96w PLATE, SILICONE MAT or GYUTO BEAD during the process?
No polymer contamination has been observed by LC-MS of tryptic digests of samples, which were prepared with the BeatBox Tissue kit 96x in combination with the iST family of sample preparation kits. The GYUTO BEAD has a special bead-coating to prevent protein binding during sample preparation and the SILICONE MAT and PLATE are resistant to the ingredients of the LYSE buffer, thus eliminating the risk of any contamination/leaching occurring from these sources.
11.29. How many times can you run a BeatBox 96w PLATE?
The number of samples per run can be freely selected by the user, e.g. 1x 96 samples, or 4x 24 samples. However, each well and GYUTO BEAD can be used only once, and it is recommended not to run the BeatBox 96w PLATE more than 40 minutes in total (independent of the setting).
NOTE: Do not remove the GYUTO BEADS from used wells; transfer only the homogenate/lysate for downstream steps.
NOTE: Do not remove the GYUTO BEADS from used wells; transfer only the homogenate/lysate for downstream steps.
11.30. Can I run the homogenization process multiple times if my sample still contains visible tissue pieces after a 10 min run?
You can run the BeatBox 96w PLATE with samples multiple times, but it is not recommended to exceed 40 minutes in total (independent of the settings). The number of runs depends on the hardness of the tissue.
NOTE: You can increase the power of homogenization in the additional runs. E.g., from STANDARD to HIGH setting.
NOTE: You can increase the power of homogenization in the additional runs. E.g., from STANDARD to HIGH setting.
11.31. Can I use the homogenate straight with the PreOmics’ kits?
Yes, there is no need to centrifuge the homogenate. PreOmics’ kits are designed to work with tissue homogenate/cell lysate. Before proceeding with PreOmics’ DIGEST step, estimate the protein content of the homogenate and transfer only the volume that contains approx. 100 µg of protein.
NOTE 1: For protein quantitation assays use only clear supernatant. Take a homogenate aliquot and centrifuge with >10 000 xg for 15 mins to remove cell or tissue debris.
NOTE 2: If you are using your own lysis buffer, you can also continue with PreOmics kits (subject to composition compatibility – for recommendations and limitations, see FAQ 1.1).
NOTE 1: For protein quantitation assays use only clear supernatant. Take a homogenate aliquot and centrifuge with >10 000 xg for 15 mins to remove cell or tissue debris.
NOTE 2: If you are using your own lysis buffer, you can also continue with PreOmics kits (subject to composition compatibility – for recommendations and limitations, see FAQ 1.1).
11.32. Can I freeze the TUBES of the BeatBox Tissue Kit 24x with the GYUTO BEAD and my sample?
Yes, the BeatBox Tubes can be frozen (-20 °C and -80 °C) with tissue samples, with or without lysis buffer, and before or after homogenization. Please follow the instructions for use for Eppendorf Safe-Lock Tubes and do not use the tubes with liquid nitrogen.
NOTE: PreOmics’ iST LYSE buffer is incompatible with freezing of tissue homogenates/cell lysates in TUBES due to increased protein amounts. As alternative, we recommend using RIPA buffer or SP3 LYSE for lysis and subsequent storage.
NOTE: PreOmics’ iST LYSE buffer is incompatible with freezing of tissue homogenates/cell lysates in TUBES due to increased protein amounts. As alternative, we recommend using RIPA buffer or SP3 LYSE for lysis and subsequent storage.
11.33. Can I remove the GYUTO BEAD from the BeatBox 96w PLATE after BEATBOX homogenization to move to the next sample preparation step?
Yes, it is possible to remove all 96 GYUTO BEADS simultaneously with help of the BeatBox bead remover. Please contact us for more details via info@preomics.com. If you only want to process a few samples, we recommend instead to transfer the homogenate from the BeatBox 96w PLATE to a fresh sample vessel.
11.34. Can I keep the GYUTO BEAD in the well of the 96w PLATE with homogenate after BeatBox homogenization to move to the next sample preparation step?
In principle, the GYUTO BEAD can be present in the homogenate for further sample preparation, such as digestion. However, please keep in mind that the max. well volume of 150 µL is small and incompatible with the PreOmics’ kit workflows. For practical reasons, we therefore recommend transferring the homogenate/lysate to a fresh sample vessel before continuing with the next sample prep step.
11.35. Can I use 0.2/0.5 mL tubes instead of the BeatBox 96w PLATE?
The BeatBox plate adapter is only compatible with the 96w PLATE and not with single 0.2/0.5 mL tubes. Certain types of 0.5 mL tubes are compatible with the BeatBox tube adapter and certain types of 0.2 mL tubes are compatible with the BeatBox plate adapter when placed into a suitable tube rack. Please contact us via info@preomics.com for more information. However, PreOmics will not support any issues with possible cross-contamination, leaching or loss of samples, when switching to different tubes or when transferring the GYUTO BEAD.
11.36. How can I reduce the sample viscosity after BeatBox homogenization?
Increased sample viscosity after tissue homogenization or cell lysis is often caused by DNA released from the nuclei. Since BeatBox only minimally shears DNA, we recommend to break down DNA through addition of nucleases or sonication. This should reduce sample viscosity.
Nucleases like Benzonase are compatible with all PreOmics’ LYSE buffers. Please mix the enzyme directly with the LYSE buffer, add the mix to your samples and continue with sample homogenization in the BeatBox. For further recommendations on nuclease amounts, see FAQ 1.25.
Nucleases like Benzonase are compatible with all PreOmics’ LYSE buffers. Please mix the enzyme directly with the LYSE buffer, add the mix to your samples and continue with sample homogenization in the BeatBox. For further recommendations on nuclease amounts, see FAQ 1.25.
11.37. Is the BeatBox bead remover compatible with plates that are not the BeatBox 96w PLATE?
The BeatBox bead remover is only compatible with the BeatBox 96w PLATE and the corresponding GYUTO BEAD COLLECTION RACK from the accessory box. We do not recommend using it together with other plates.
11.38. Is polymer released from the BeatBox bead remover foil during the process?
No polymer contamination has been observed by LC-MS of tryptic peptides and corresponding buffer controls. Samples were prepared with the BeatBox 96w kit, followed by removal of the GYUTO BEADS with the BeatBox bead remover and bead remover foil, and digestion and peptide clean-up with the iST family of sample preparation kits. The BeatBox bead remover foil is a single-use consumable that is resistant to the ingredients of the LYSE buffer, thus eliminating the risk of any contamination/leaching occurring from these sources.
11.39. Is the BeatBox bead remover compatible with other plastic films or foils?
This is not recommended. PreOmics will not support any issues with possible cross-contamination or leaching when switching to a different film or foil. The supplied BeatBox bead remover foil is optimized to work with the BeatBox bead remover. Other less elastic films or foils are likely pierced during the removal procedure, leading to contamination of the tool.
11.40. How can I clean the BeatBox bead remover, BeatBox plate adapter, and tube adapter?
In case of contamination of these items with sample, you can clean them by disinfectant wipe down using a damp cloth with the following substances: 70% ethanol, 70% isopropanol, 10% sodium hypochlorite, aldehyde-based disinfectants. Please refer to the BeatBox user manual for details.
12. FFPE sample processing with BeatBox and iST
12.1. How do you avoid issues with static charge when transferring FFPE tissue into the BeatBox 96w PLATE?
Please pre-fill the wells of the BeatBox Tissue Kit 96x plate with lysis buffer, then add the FFPE tissue. From our experience, this reduces static charge.
12.2. Which types of FFPE tissue can be processed with the BeatBox/iST FFPE workflow?
The BeatBox/iST FFPE protocol was developed for scrolls/sections with a thickness of 10-20 µm. The advantage of the workflow is that no separate xylene-based deparaffinization step is necessary. However, deparaffinized FFPE tissue can likewise be used as starting material.
Micro-/macrodissected specimen or thinner scrolls are also compatible as long as the amount of extracted protein is 1 µg or higher. This is the lower input limit for the iST CARTRIDGE. For low protein input amounts of 1-20 µg, please contact us via info@preomics.com for a modified protocol.
Punches are more difficult to homogenize and factors like punch size, compactness and type of tissue will influence the success of homogenization. Please keep the following aspects in mind:- FFPE tissue is dehydrated and thus contains a higher protein concentration than the corresponding fresh-frozen punch of the same size. Therefore, the punch size needs to be adjusted experimentally, as to not exceed the max. input of the BeatBox 96w PLATE (500 µg protein or 5 mg tissue wet weight).- If possible, cut the punches into smaller pieces.- To support the homogenization process of the tissue punch, it is recommended to perform multiple rounds/cycles of BeatBox homogenization and de-crosslinking at 80-95 °C ( e.g. 10 min BeatBox 30-60 min heating -> 10 min BeatBox -> 30-60 min heating).
Micro-/macrodissected specimen or thinner scrolls are also compatible as long as the amount of extracted protein is 1 µg or higher. This is the lower input limit for the iST CARTRIDGE. For low protein input amounts of 1-20 µg, please contact us via info@preomics.com for a modified protocol.
Punches are more difficult to homogenize and factors like punch size, compactness and type of tissue will influence the success of homogenization. Please keep the following aspects in mind:- FFPE tissue is dehydrated and thus contains a higher protein concentration than the corresponding fresh-frozen punch of the same size. Therefore, the punch size needs to be adjusted experimentally, as to not exceed the max. input of the BeatBox 96w PLATE (500 µg protein or 5 mg tissue wet weight).- If possible, cut the punches into smaller pieces.- To support the homogenization process of the tissue punch, it is recommended to perform multiple rounds/cycles of BeatBox homogenization and de-crosslinking at 80-95 °C ( e.g. 10 min BeatBox 30-60 min heating -> 10 min BeatBox -> 30-60 min heating).
12.3. What is the peptide yield per FFPE scroll?
Peptide yields highly depend on the area of the tissue embedded in the paraffin block, the thickness of the slice/scroll, and on the tissue type. For example, we obtained 5-8 µg peptide out of 7 mm²/10 µm scroll of mouse cardiac muscle, but up to 180 µg peptide out of 140 mm²/10 µm scroll of mouse liver. The dimensions of the embedding cassettes /paraffin blocks were similar in both cases.
Exemplary peptide yields from FFPE tissue:
Exemplary peptide yields from FFPE tissue:
Tissue type
Tissue volume in scroll (mm3)
Peptide yield (NanoDrop)
Mouse kidney
0.78
46-68 µg
Mouse liver
1.43
88-184 µg
Mouse cardiac muscle
0.37
28-45 µg
Mouse cardiac muscle
0.07
5-8 µg
12.4. How does the BeatBox/iST workflow remove paraffin from the FFPE curl/scroll ?
The homogenate obtained with BeatBox contains finely dispersed paraffin, which melts during the subsequent heating step. During cooldown, parts of the paraffin will separate from the aqueous homogenate in form of a paraffin ring above the liquid. The paraffin ring will not interfere with further processing and should be left in the BeatBox 96w PLATE when transferring the homogenate.
The homogenate will still contain paraffin and often appears milky white. After the DIGEST step, this residual paraffin is transferred with the sample to the iST CARTRIDGE and subsequently removed when applying WASH 0.
The homogenate will still contain paraffin and often appears milky white. After the DIGEST step, this residual paraffin is transferred with the sample to the iST CARTRIDGE and subsequently removed when applying WASH 0.
12.5. What is the maximum FFPE tissue input that can be processed with the BeatBox/iST FFPE protocol?
With the BeatBox Tissue Kit 96x you can process full curls/scrolls/sections of up to 20 µm thickness (or 2x10 µm). Thicker slices do not fit into the well. For 20 µm curls/scrolls/sections, increase the lysis buffer volume to 100 µL.
The type and size of the tissue embedded in the paraffin block greatly influences the protein yield per curl/scroll/section. For the subsequent iST workflow, max. 100 µg protein should be loaded onto the iST CARTRIDGE to ensure efficient peptide clean-up.
If you want to process punches, please see FAQ 12.2. for further information.
The type and size of the tissue embedded in the paraffin block greatly influences the protein yield per curl/scroll/section. For the subsequent iST workflow, max. 100 µg protein should be loaded onto the iST CARTRIDGE to ensure efficient peptide clean-up.
If you want to process punches, please see FAQ 12.2. for further information.
12.6. What is the smallest FFPE tissue size that can be processed with the BeatBox/iST FFPE protocol?
See FAQ 12.2.
12.7. What’s the proteomic depth one can achieve with the BeatBox/iST FFPE workflow?
The achievable proteomic depth depends on many different factors, for example on the area of the tissue embedded in the paraffin block, the thickness of the slice/scroll, and on the tissue type. The LC-MS setup and method will also influence the results. Please refer to our application notes for exemplary data or contact us at info@preomics.com.
12.8. How can I determine the protein yield of my FFPE homogenate?
When PreOmics LYSE buffer is used, we recommend using the Pierce™ BCA Protein Assay Kit - Reducing Agent Compatible (Thermo Fisher, cat.no. 23250 / 23252).
Vortex the supernatant of homogenized FFPE sample. Take ~10 µL and dilute to stay within linear range. Since protein yields highly depend on the area of the tissue embedded in the paraffin block, the thickness of the slice/scroll, and on the tissue type, we cannot provide concrete dilution factors. For orientation, we used 1:3 for mouse heart tissue and 1:5 for mouse liver tissue of 0.5 to 1 cm diameter in 10 µm curls.Perform the assay as described by manufacturer.
Vortex the supernatant of homogenized FFPE sample. Take ~10 µL and dilute to stay within linear range. Since protein yields highly depend on the area of the tissue embedded in the paraffin block, the thickness of the slice/scroll, and on the tissue type, we cannot provide concrete dilution factors. For orientation, we used 1:3 for mouse heart tissue and 1:5 for mouse liver tissue of 0.5 to 1 cm diameter in 10 µm curls.Perform the assay as described by manufacturer.
12.9. Which temperature should I use for the de-crosslinking step in the BeatBox/iST FFPE protocol?
The protocol specifies a temperature range of 80-95 °C and temperatures at the upper end should increase extraction and de-crosslinking efficiency.
If you use cap strips to seal the BeatBox 96w PLATE and a thermoshaker for heating, please note the following: The temperature reached inside the wells may vary between different thermoshaker models. At very high temperatures the cap strip may burst open due to high vapor pressure of the lysis buffer. To avoid sample loss, perform a test run with lysis buffer to identify the highest possible temperature for your thermoshaker setup. We do not recommend using a heated lid (e.g. ThermoTop # 5308000003 Eppendorf). Use a conventional lid instead. If only a ThermoTop is available, reduce the temperature to 80-85°C.
A lower heating temperature may sometimes lead to lower protein extraction efficiency. To improve results, you can perform a second cycle of BeatBox and heating.
Alternatively, use a PCR cycler with a heated lid exerting pressure from the top and close the BeatBox 96w PLATE with the SILICONE MAT. This allows you to select a higher temperature for the de-crosslinking step.
If you use cap strips to seal the BeatBox 96w PLATE and a thermoshaker for heating, please note the following: The temperature reached inside the wells may vary between different thermoshaker models. At very high temperatures the cap strip may burst open due to high vapor pressure of the lysis buffer. To avoid sample loss, perform a test run with lysis buffer to identify the highest possible temperature for your thermoshaker setup. We do not recommend using a heated lid (e.g. ThermoTop # 5308000003 Eppendorf). Use a conventional lid instead. If only a ThermoTop is available, reduce the temperature to 80-85°C.
A lower heating temperature may sometimes lead to lower protein extraction efficiency. To improve results, you can perform a second cycle of BeatBox and heating.
Alternatively, use a PCR cycler with a heated lid exerting pressure from the top and close the BeatBox 96w PLATE with the SILICONE MAT. This allows you to select a higher temperature for the de-crosslinking step.
12.10. My FFPE tissue is very rigid and challenging to homogenize. Is there a special recommendation?
Depending on the amount of input material, you may have to perform multiple rounds of BeatBox and heating to help with homogenization/hydration of the tissue. We recommend testing the following sequence: 10 min BeatBox -> 30 min at 80-95 °C -> 10 min BeatBox -> 30 min at 80-95 °C. If the tissue is still not fully homogenized, continue with another cycle of BeatBox and heating.
12.11. Can I freeze the leftover homogenate from FFPE tissue?
Yes, this is possible. FFPE tissue homogenates in iST LYSE or SP3 LYSE can be frozen to be processed at a later timepoint. Just note that if you homogenized paraffin containing FFPE samples, the paraffin dispersed in the solution will precipitate. After thawing the sample, please heat to >60°C until the paraffin is dissolved.
NOTE: For freezing, please follow the instructions for use for Eppendorf Safe-Lock Tubes and do not use the tubes with liquid nitrogen.
NOTE: For freezing, please follow the instructions for use for Eppendorf Safe-Lock Tubes and do not use the tubes with liquid nitrogen.
12.12. Is the FFPE workflow also compatible with the BeatBox Tissue Kit 24x?
We currently do not recommend using the BeatBox Tissue Kit 24x.
12.13. Can the BeatBox/iST FFPE workflow be combined with the SP3-iST Add-on?
Yes, this is possible. We recommend replacing the acidic SP3 LYSE supplied with the kit with a different lysis buffer with a higher pH, e.g. 50 mM TEAB (pH 8.5) with 5% SDS or RIPA buffer. Lysis buffers with basic pH typically show better extraction performance with FFPE tissue.For a modified protocol, please contact us at info@preomics.com
12.14. Are SDS-based lysis buffers also compatible with your BeatBox/iST FFPE workflow?
Yes, these buffers can also be used for the protein extraction step. If the SDS concentration in the sample is 0.1%, the iST workflow remains unchanged. For higher amounts of SDS, use the SP3-iST Add-on kit to remove SDS before continuing with the DIGEST step of the iST workflow. After protein clean-up on the SP3-beads, residual paraffin can still be still present in the sample. We recommend using WASH 0 for full paraffin removal during iST clean-up, as outlined in the FFPE protocol.
13. Accessories for PreOmics technologies
13.1. What is the Metal Heating Shaker Adapter?
The Metal Heating Shaker Adapter guarantees optimal heat transfer for our CARTRIDGES compared to planar heating systems. It is compatible with any heating shaker in the SPSS format and many liquid handling platforms. It is also directly compatible with our 96-well CARTRIDGE adapter plates.
13.2. When purchasing the PreOmics’ 96 reaction kits, do I need to order the 96-well adapter plate too?
All our kits in the 96 reaction format already contain the 96-well adapter plate required for convenient handling of larger sample numbers.
14. Ordering, Shipping & Storage
14.1. How can I order your products?
Customers from North America please order via PreOmics Inc. (USA), customers from the rest of the world please order via PreOmics GmbH (Germany)
We offer several options to order our products:
- Send an email to: order@preomics.com
- Call us: +49-89-2314-163-0
- Fax us: +49-89-2314-163-99
We offer several options to order our products:
- Send an email to: order@preomics.com
- Call us: +49-89-2314-163-0
- Fax us: +49-89-2314-163-99
14.2. Does PreOmics ship worldwide?
Yes, we do ship our products worldwide. For the convenience of our US customers, we ship from our warehouse in New Jersey.
14.3. Is it possible to order individual items from your kits?
We provide complete solutions to ensure best results for your LC-MS/MS analyses. Adaptions to our lysis buffers might be necessary though for specific experimental questions. Upon request, we provide the appropriate 2x concentrated LYSE buffer and the WASH0 for use with plant, algae and diatom samples.
14.4. How do you ship your products and how shall I store them?
We ship at ambient temperature. Upon arrival, please store the lyophilized enzyme mix (red DIGESTtubes) at -20°C and the rest of the kit at room temperature.
14.5. Can I freeze whole kits upon arrival?
No, freezing is detrimental to our buffers. Only the lyophilized enzymes (red DIGEST tubes) in the iST, iST-REG-PSI, iST-NHS and iST-BCT kits should be frozen upon arrival for long-term storage.
14.6. How do I store resuspended DIGEST in case I have leftover solution?
Resuspended DIGEST can be stored at 4°C for up to two weeks. For long-term storage, lyophilize the DIGEST again, lyophilized DIGEST can be stored at -20°C for up to nine months.
14.7. How long can I store the PreOmics’ kits?
We guarantee a minimum remaining shelf life of three months after receiving our products. Please refer to the shelf life information printed on each kit box for further details.
14.8. Can I still use a kit after its shelf life has expired?
The performance of our LYSE / LYSE-NHS / LYSE-BCT will drop significantly after the expiration date. Thus, we do not recommend to use our kits after the shelf life has expired.