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iST technology for sample preparation

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 use the SP3-iST kit. (see protocol here).

Please refer to the following table or contact us to check the compatibility of your lysis buffer with the iST system:
Own lysis buffer
PreOmics compatible?
Max. volume lysis buffer
25 µL
dilute with equal volume of appropriate 2X concentrated LYSE reagent
RIPA (max. 0.1% SDS, 1%SDC, 1% Triton x-100)
25 µL
dilute with equal volume of appropriate 2X concentrated LYSE reagent
Urea (max. 2M; no thiourea)
25 µL
dilute with equal volume of appropriate 2X concentrated LYSE reagent
perform protein precipitation
High salt (e.g. >0.5M NaCl)
perform protein precipitation
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 µLsample + 11 µL2X 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.
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 min
8. 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
Can I add protease inhibitors to my sample?
Although protease inhibitors are in general compatible with our iST protocols, we recommend not to add additional exogeneous proteins in order to not contaminate your sample.
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.
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 "Shall I adjust the buffer volumes depending on the protein starting amounts?"
What is the maximum volume I can load on the PreOmics’ CARTRIDGES?
The maximum volume is 250 µL.
Which protein quantification methods are compatible with the PreOmics' kits?
Most classical assays are compatible with our PreOmics’ iST, iST-NHS and iST-BCT LYSE buffers*. We recommend the BCA assay or the tryptophan quantification method. Some assays require dilution with distilled water to achieve the best results shown below:

- BCA: none
- microBCA: 1:100 with PBS
- Coomassie: 1:20
- Lowry: 1:4
- Tryptophan: none

*BCA assay is NOT compatible with SP3-LYSE, please seefor more information
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 start-ing 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.
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).
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.
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 "Which lysis temperature shall I use?".
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.
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.
How can I automate my sample processing efforts?
The PreOmics' PreON isa dedicated sample preparation platform suitable for 12 or 16 samples, see here or click on the "PreON platform" tab for more information. PreOmics is working with liquid handling systems providers and application notes are available here or we are happy to provide technical assistance to transfer the PreOmics methodology onto your existing platform if appropriate.
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.
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 "Are the PreOmics kits compatible with absolute quantification?"). Our peptide recovery is typically >80% for 1 - 100 µg protein starting material.
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.
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 reaction: 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 reaction: 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 reaction: 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 reaction: 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
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.
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.
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.
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.
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.

Sample type recommendations

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 "Shall I adjust the buffer volumes depending on the protein starting amounts?"). 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.
Starting amount
Protein amount
Mammalian Cell Line (e.g. HeLa)
6E5 cells
100 µg
Yeast (S.cerevisae)
OD600 = 0.6
100 µg
Bacteria (E.coli)
OD600 = 0.5
100 µg
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 mg / 100 mg
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.
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 "How do I perform protein precipitation?") and using the resulting pellet as input material for the iST-BCT/iST-NHS protocol.
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 "Which protein quantification methods are compatible with the PreOmics’ kits?"). For highly concentrated CSF samples, follow the recommendations for non-depleted plasma/serum (see "How much raw material do I need for the PreOmics’ kits?") For diluted CSF samples (volume larger than 25 µL), perform protein precipitation (see "How do I perform protein precipitation?") 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.
How do I process urine samples?
Protein concentrations in urine samples vary substantially, we recommend either concentrating or precipitating the protein (see "How do I perform protein precipitation?") 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.
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.
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.
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 (see 1.5.). PreOmics’ BeatBox instrument efficiently and reproducibly homogenizes tissues and lyses cells from various sources. For more information, see https://www.preomics.com/beatbox.
How do I process FFPE samples?
Processing of FFPE samples requires de-paraffinization of the FFPE punches/slides. In addition, deparaffinized samples require a longer heat treatment for efficient de-crosslinking: increase the initial heating step from 10 to 60 min at 95°C. More details can be found in the FFPE protocol or use the SP3-iST kit (protocols available here).
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.
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).
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).

Chemical labeling (iTRAQTM, TMTTM)

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
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.
Shall 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.
When shall 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 split equally on different CARTRIDGES (binding capacity of 100 µg per CARTRIDGE).
B. Perform workflow until end of vacuum concentrator step. Resuspend individual channels in LC-LOAD and then pool channels accordingly.
We advise to measure the peptide concentration of each channel for optimal channel pooling results.

iST-PSI for positive pressure processing

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).
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.
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.

Peptide Cleanup

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”.
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
Sample types
urine, plants
lipids, polymers, detergents
hydrophobic contaminations
hydrophilic contaminations
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).
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.
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.
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.
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).
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.

Phoenix kit

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.
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.

iST-Fractionation Add-on

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.
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.
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).
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.

SP3-iST Add-on kit

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.
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 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)
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
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 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
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.
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.
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.
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
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:
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
How do I prepare tissue or FFPE samples with the SP3-iST kit?
We recommend homogenizing the sample in a sonicator with glass beads. Add 40-50 mg glassbeads and 50 µLSP3 LYSE to sample and make up to 100 µL with RESUSPEND. Sonicate for at least 10 cycles; 30 sec ON/OFF. Then heat for at least 10 minutes at 95 °C with shaking if possible at 1000- 1400 rpm. For tougher tissue like heart or muscle, repeat sonication and heating step a second time.
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).

PreON automation platform

What are the 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.) x62 cm (24.4 in.) x 86 cm (34.0 in.) with a weight of 71.5 kg (157.6 lbs).
Which sample types can be processed on the PreON?
The PreON can process protein pellets, intact cells, body fluids and tissue lysates.
How many samples can I process on the PreON per day?
The sample throughput is 12 samples per run with the option to execute multiple runs per day (16 position PreON is available for use with TMTpro). For iST workflows, up to three runs per day to give a total of 36 samples are feasible. For iST-NHS workflows, up to two runs per day to give a total of 24 samples are feasible.
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.
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 will cause pipetting errors.
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.
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. Once every six months we recommend to perform a tightness test for the pipetting unit (all required utensils provided).
How is the PreON serviced?
Our global and trusted partner PEAK-Service is the leading global provider for technical services for medical, analytical and industrial equipment. Contact us to inquire about further servicing options and pricing.

BeatBox instrument

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.
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.
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.

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.
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 model organisms, e.g. human, rat, mouse, plant.

NOTE: For plant samples please contact info@preomics.com
Have you tested bone or fingernail?
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.
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.
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.
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 runs for complete homogenization.
Can I process FFPE tissues with the BeatBox?
For information, please contact info@preomics.com
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. 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).

If you continue with PreOmics kits, be aware that after the homogenization step, your lysis buffer will be mixed with PreOmics’ LYSE buffer, based on recommendations in FAQ 1.1.
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.

NOTE: PreOmics LYSE buffer’s efficiency to lyse, reduce and alkylate sample rapidly decreases when used below room temperature.
Can I centrifuge the BeatBox 96w PLATE with GYUTO BEADS after homogenization?
Yes, you can centrifuge the BeatBox 96w PLATE after homogenization. We recommend quick spin down of the BeatBox 96w PLATE at 300-500 xg for 30-60 sec before removing the SILICONE MAT to avoid cross-contamination. If high concentrations of SDS (up to 5%) 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.
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 the BeatBox Tissue Kit 24x, spin down the BeatBox TUBES (1500 xg, 30-60 sec) after homogenization and transfer the homogenate using a pipet.
What’s the size of the GYUTO BEAD?
This is proprietary information that cannot be shared.
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.
Does the BeatBox have a heating/cooling function?
The BeatBox does not have heating or cooling options. The BeatBox operates at room temperature.
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.
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 be stable at room temperature during the homogenization process.

NOTE: Nuclease/protease/phosphates cocktails can be added into the PreOmics’ LYSE if needed.
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 60 mins. Please, use an additional TRANSPARENT LID above the BeatBox 96w PLATE and SILICONE MAT to prevent SILICONE MAT peeling caused by temperature. NOTE: Spin down the BeatBox 96w PLATE before removing SILICONE MAT and TRANSPARENT LID to prevent cross-contamination. Recommended speed is 500 xg for 30-60 sec.
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 homogenization occurs as a result of the chaotic, nondirected movement of a single magnetic GYUTO BEAD. The only moving part of the BeatBox is the GYUTO BEAD in the plate well or tube.
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 runs and/or the high setting for complete homogenization.
Can we use plates that are not the BeatBox 96w PLATE?
This is not recommended; PreOmics will not support any issues with possible cross-contamination, leaching or loss of samples, when using different plates. The supplied plate is optimized to work with the GYUTO BEAD. Homogenization efficiency can be affected if the plate used does not have the correct dimensions.
Can I use 1.5 mL tubes instead of the 2 mL tubes provided with the BeatBox Tissue Kit 24x?
This is not recommended for two reasons: Firstly, the GYUTO BEAD might get contaminated during transfer from the 2 mL to the 1.5 mL tube. Secondly, the ratios between the GYUTO BEAD : volume : well shape/size is critical to efficient homogenization and have been optimized for the 2 mL tube supplied with the PreOmics kit.
Can I use my own 2 mL tubes instead of the supplied BeatBox TUBES?
This is not recommended; PreOmics will not support any issues with possible cross-contamination, leaching or loss of samples, when switching to different tubes.
Can I transfer GYUTO BEADS to an in-house 96 well plate, if it is similar to the BeatBox Tissue Kit 96x one?
This is not recommended due to potential GYUTO BEAD contamination, as well as the potential issues outlined above for use of any plate that is not the supplied BeatBox 96w PLATE. The ratios between the GYUTO BEAD : volume : well shape/size is critical to efficient homogenization and although another plate maybe appear to be similar, individual dimensions may not be.
Would it be possible to use the BeatBox for genomics, metabolomics, lipidomics and other omics applications?
The BeatBox has been designed primarily for proteomics applications. Every part of the kit is compatible with general lysis buffers used for proteomics applications. For other omics applications, aqueous buffers with maximum of 5% SDS are recommended. For further information, please contact us at info@preomics.com.
Is it possibile 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?
Please contact us for more details via info@preomics.com.
Which protein quantification method you would recommend using after BeatBox homogenization?
When PreOmics LYSE buffer is used, we recommend the BCA assay or the tryptophan quantification method. 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.
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.
What is moving in the BeatBox during the homogenization process?
The only thing that moves during the process is the GYUTO BEAD.
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.
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.

NOTE: DO NOT remove the GYUTO beads from used wells; transfer only the homogenate/lysate for downstream steps.
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.
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: 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.
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 ºC and -80 ºC) with tissue samples, with or without lysis buffer, and before or after homogenization.

NOTE: PreOmics’ 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 for lysis and subsequent storage.
Can I remove the GYUTO BEAD from the BeatBox 96w PLATE after BEATBOX homogenization to move to the next sample preparation step?
This is not recommended, due to possible cross-contamination. Instead, we highly recommend transferring the homogenate from the BeatBox 96w PLATE to a fresh sample vessel.
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.
Can I use 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 0.5 mL tubes.

Accessories for PreOmics technologies

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.
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.

Ordering, Shipping & Storage

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 severaloptions 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
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.
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.
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.
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.
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.
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.
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.

Any more questions?

Don’t hesitate, contact us!