- Most classical assays are compatible with our LYSE and LYSE-NHS buffers. We recommend the BCA assay or the tryptophan quantification method. Some assays require dilution with destilled water to achieve best results.
- BCA: none
- Bradford: 1:4
- Coomassie: 1:20
- Lowry: 1:4
- Tryptophan: none
- We recommend storage at RT for up to 9 months. The iST and iST-NHS kits are shipped at ambient temperature. Upon arrival, please store the enzyme mix at -20°C and the rest of the kit at room temperature.
- We get highly reproducible results with protein starting amounts ranging from 1 µg to 100 µg. For low input information please see “Should I adjust the volumes of the buffers with protein starting amounts?”
- Please inquire for further information on the starting amounts for various sample types. A short overview of recommended raw material amounts is also given on the inside of the iST and iST-NHS kits and can be downloaded also on our resources page.
- We strongly recommend to use the LYSE or LYSE-NHS buffers provided with the iST or iST-NHS kits, respectively. Sonication (e.g. Diagenode Bioruptor systems) can be used together with the LYSE and LYSE-NHS buffers to increase cell lysis.
If you want to use your own lysis buffer, the efficiency of cell lysis and thus protein identification will likely suffer. Please refer to the following table or contact us to check the compatibility of your lysis buffer with the iST system:
For further information on concentrated LYSE or LYSE-NHS buffers contact us.
|own lysis buffer||compatible with LYSE/LYSE-NHS||max. volume own lysis buffer||remarks|
|RIPA (max. 0.1% SDS, 1% SDC, 1% Triton X-100)||Yes||25 µL||dilute with 25 µL 2X concentrated LYSE|
|PBS||Yes||25 µL||dilute with 25 µL 2X concentrated LYSE|
- For simplicity reasons we recommend usage of buffer volumes indicated in the protocol. You can adjust the volume of LYSE and DIGEST to your starting amounts (e.g.: 20 ug protein starting material, 10 ul LYSE, 10 ul DIGEST, keep the other buffers as indicated in the protocol). For less than 20ug protein starting material, stay with 10ul LYSE and DIGEST, respectively. Accordingly, you may adjust the volumes for chemical labelling and quenching as recommended by the label manufacturer.
- We recommend to perform the lysis/denaturation at 95°C and only use lower temperatures for temperature-sensitive samples such as IP samples. We have tested lower temperatures down to 60°C and do not see any differences in parameters tested (IDs, alkylation rates, etc.).
- Yes, IP samples can directly be processed with the kit. However, the protocols for Agarose- or magnetic affinity matrices slightly differ, please inquire or refer to our Resources for adapted protocols.
- We recommend to perform your standard homogenization in our lysis buffer (e.g. bead milling, grinder, etc.). After homogenization boil the sample (FFPE samples 1h at 95°C) and continue with the protocol as recommended. If you need additional LYSE buffer please contact us.
- Yes, FFPE samples can directly be processed with the kit. Please inquire or refer to our website for an adapted protocol.
- Yes, urine samples can directly be processed with the kit. However, given the high amount of bilirubin, we provide an extra washing buffer for urine samples. Please inquire or refer to our website for an adapted protocol.
- Our LYSE and LYSE-NHS buffers denature, reduce and alkylate proteins. Thus, please consider the following fixed modifications in your database search:
|LYSE-NHS||Alkylation||Specific cysteine modification||C6H11NO||[C]||+113.084 Da|
- The iST and iST-NHS Kits 96 rxn allow you to start immediately with ready-to-go reagents and offer the flexibility to process either 96 samples at once or multiple batches of smaller sample numbers.
- Chemical labeling experiments require very high peptide labeling efficiencies (>98%) for proper quantification. When struggling with lower labeling efficiencies, please see the following recommendations:
- Make sure that the sample input material (e.g. cell pellet) is not contaminated with residual buffers or cell culture media containing primary amines that interfere with chemical labeling.
- We recommend to use only fresh labeling reagents. 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 labeling agent 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.
- Currently, our kits come with an enzyme mix consisting of LysC and trypsin. We are developing solutions for alternative enzymes.