I am searching for a protein lysis buffer for human cancer cells that is compatabile with mass spec. analysis.I have used RIPA buffer but the people from the mass spec facility told me, that the lysis buffer must not contain: SDS, Triton, NP-40 or Tween.
We recommend to use your regular lysis buffer but to replace SDS with n-dodecyl maltoside (DDM).
While you can get rid off SDS (e.g. by precipitation), even small contaminations have an impact on your ionization efficiency. DDM has much less ion suppression effects than SDS, plus it elutes in one peak at the end of the chromatogram.
We also recommend not to use any Triton, NP-40 or Tween. Just leave it out for your proteomics analysis.
We use 7M Urea/ 2M Thioures/ 2% N-octylglucoside, or If you have to use SDS or other kind of detergents you can follow the FASP (Mann's lab) protocol. In our hands works 10/10.
I don't think there is a problem in using SDS in your buffer preparation, as you can always use S-TRAP columns to clear out the SDS. I regularly lyse my cells in 5 % SDS and have good MS chromatograms.
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biochemical Techniques
- Analytical Chemistry
- pH