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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.

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).

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:

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.

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.

See FAQ 12.2.

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.

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.

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.

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.

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.

We currently do not recommend using the BeatBox Tissue Kit 24x.

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

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.

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.

All our kits in the 96 reaction format already contain the 96-well adapter plate required for convenient handling of larger sample numbers.

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

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Yes, we do ship our products worldwide. For the convenience of our US customers, we ship from our warehouse in New Jersey.

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.

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.

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.

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.

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.

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.