Hi ,
I need to do phosphoprotomics on GFP pull-downs and total lysates using TMT labbeling and I am hesitating about the best buffer conditions. Does anyone have experience on GFP-trap followed by TMT labbeling? Which buffer would you recommend?
Is Ambic buffer compatible with TMT labeling or should I use HEPES instead? My lysis buffer will be HEPES/Triton0.5%/EDTA/NaCl for the input samples.
Thank you!
Dear Eugenia,
As the TMT labeling reagents are activated esters which are intended to label amino groups (lysines), they will react with anything else that has a nucleophilic nitrogen: Ammonium salts, tris, etc. You need to do a very efficient buffer exchange (gel filtration/desalting) or dialysis to remove any of these interfering components, once they are in. HEPES and any other slightly alkaline buffers (pH 8) should work, though, as long as they don't contain amino groups.
Dear Wolfgang,
Thank you for your answer! I will go with HEPES to resuspend the beads thenz I already substituted the tris in my lysis buffer for HEPES for the same reason!
I am still trying to find some references where they use gfp-trap combined with tmt labeling and proteomics or phosphoproteomics, and I am not finding it so I news to design my own protocol (fingers crossed!). If someone has a successful protocol, I would appreciate it a lot!
Best wishes,
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