Problem with removing fusion protein. MALDI TOF. Tricine SDS PAGE.
Posted 26 March 2012 - 08:07 AM
I have problem with cleave a peptide (3,2 kDa) off a fusion protein.The construct contains protease site between fusion protein and the peptide of interest (actually I have 2 constructs: with enterokinase and with factor Xa site). Both proteases seem to work, but both show some nonspecificity. I use tricine sds gel, that is for visualising small peptides, but I don’t get a band at the needed position. The same result is with MALDI TOF. Maybe the concentratoin of product is too low to visualise it on gel, but I geally worry about MALDI result because it shows nothing at 3,2 kDa.. Honestly it barely shows something... I guess any contaminant may interrupt. Probes contain 1 M urea, 20-50 mM Tris, 1- 2 mM CaCl2, 0,1% Tween20, 100 mM NaCl and some imidasole.. Can any of this reagents interrupt MALDI? Or may you share some experience about tricine gel preparation which could help me?
P.S. There is no problem with MALDI apparatus and matrix. They do their work well.
Posted 28 March 2012 - 06:51 AM
What matrix are you currently using? I have found that some matrices are incompatible with a reconstitution solution that contains phosphate, from my experience 50% acetonitrile works as a good all around "go to" reconstitution solution.
Edited by proteaMatt, 28 March 2012 - 06:52 AM.