Protein lysis buffer for mass spec - recipe (May/11/2007 )
Hello everybody!
I am searching for a recipe for a protein lysis buffer. The lysates will be used for mass spec analysis. The people from the mass spec facility told me, that the lysis buffer must not contain: SDS, Triton, NP-40 or Tween, but may contain Octylglycoside, CHAPS or Desoxycholate. Does anyone in this forum have a corresponding buffer recipe? I searched the Internet but couldn't find anything appropriate... 
I am searching for a recipe for a protein lysis buffer. The lysates will be used for mass spec analysis. The people from the mass spec facility told me, that the lysis buffer must not contain: SDS, Triton, NP-40 or Tween, but may contain Octylglycoside, CHAPS or Desoxycholate. Does anyone in this forum have a corresponding buffer recipe? I searched the Internet but couldn't find anything appropriate...
which procedure will lie between lysis step and MS?
I am searching for a recipe for a protein lysis buffer. The lysates will be used for mass spec analysis. The people from the mass spec facility told me, that the lysis buffer must not contain: SDS, Triton, NP-40 or Tween, but may contain Octylglycoside, CHAPS or Desoxycholate. Does anyone in this forum have a corresponding buffer recipe? I searched the Internet but couldn't find anything appropriate...
Is it for MALDI or ESI? Do you want to use the lysate for MS? I´m asking, because if you´re going to use SDS-Page or FPLC before MS you could use these detergents
Are you using the direct lysate for MS right after cell lysing? I doubt it will work. I think you have to purify the desired protein first before running on MS. I am not too sure about that though.
I forgot to say: I will apply the lysate to a ubiquitin enrichment column prior to mass spectrometry. So I intend to look only at the ubiquitinated proteins. The person from the mass spec facility just told me to lyse the tissue sample without SDS, NP-40, Triton and Tween, because those would ruin the facility...
Ok, the problem is not the detergents on/in the MS, the problem is these detegents and the enrichment column. So recipe should work as lysate puffer and work on the ubiquitinated column! In my opinion the detergents gone after the enrichment step, but your MS people are afraid of "SDS, NP-40, Triton and Tween" on this column! Is that right....
The MS guy told me, that the lysates should by no means be treated with SDS,NP-40, Triton and Tween etc since they would ruin his MS facility (I will have to believe him on that, since he won't send my probes thru his MS unless I handle them as he suggests!). The protocol of the enrichment column actually allows usage of alle those detergents.
What I would like to have is a lysis buffer (for protein isolation from brain) that somehow looks like:
-PBS
-1% Octylglucopyranoside
-1% CHAPS
- protease inhibitors.
Now my question is: is this buffer obviously missing an important component? If anything comes to your mind please write! 
Dont you need a buffer component?
What type of protease inhibitors are you using? I think if EDTA, it might actually interfere with MS. Not sure about PMSF though.
What type of protease inhibitors are you using? I think if EDTA, it might actually interfere with MS. Not sure about PMSF though.
What do you mean by "buffer component"? PBS is already a buffer. Or am I barking up the wrong tree?
As to the protease inhibitor- you're right! EDTA would interfere with the MS analysis. As to PMSF....no idea...