Flag-IP elution with TEV protease - (Jun/06/2008 )
I started establishing a tandem affinitiy purification assay (TAP) in our lab. I use two FLAG peptides, followed by a TEV protease cleavage site peptide and a streptavidin binding peptide (SBP) as tags of my transcription factor. I did not succeed in eluting the proteins that bind to the FLAG agarose (M2, Sigma) with the Invitrogen TEV protease under different conditions (4°C 4h, 16°C 2h, RT 2h) as monitored by coomassie, silver staining and WB. I understand that there are several possible explanations for that (e.g. protein or tags cover the TEV cleavage site, protein undergoes degradation etc.). Do you have any good idea how to proceed now? I could circumvent the TEV cleavage step by eluting with FLAG peptide, but this would be my last solution. Thanks for every comment!
I had problems with the TEV cleavage too, I found that if the protein was not on a bead, the cleavage worked better. After I got it off the bead I used the protocol invitrogen suggests and it cleaved about 80% of the conc. protein but you really had to dilute it. Hope you can figure out a better way.