Protocol Online logo
Top : Forum Archives: : Protein and Proteomics

GST tag - expression with GST (Mar/17/2005 )

Pages: Previous 1 2 

Hello all:

Oversonication can denature proteins. I believe denatured proteins stick to everything on a column. You don't want that.

Oh, to conclude the story from my previous post, it turns out that a signicant part of the problem was the time and temperature at which we incubated the lysate with the glutathione beads. (We checked many many other things -- DNA sequence, IPTG, induction, sonication time and methods, DTT amounts in the SDS sample buffer etc etc).

We found that for our protein at least, incubating for short periods of time was better than long. A time course experiment showed that incubating for 10 minutes, 1 hour, or 3 hours enriched for our protein. But overnight incubation (which we sort of were doing before when we used a column to load) resulted in what we think is competition of some other bacterial contaminant (~60 KDa) for the glutathione beads.


QUOTE (dr_washo @ Jul 3 2005, 06:36 PM)
Oh forgot to mention, as a far as the western story is concerned that is bizarre! I'll ask my supervisor about that one and get back to you.

Dr Washo ph34r.gif

Hi Dr. Washo,

I have seen one of your replies to GST fused protein problems on SDS gel.. my GST fused protein appears different from its actual size on the SDS gel. and i know it is not Glycosylated. I have been trying to purify EYFP fused to GST and also the N-terminal EYFP , C-terminal EYFP using the GST.. the N-terminal YFP is 2/3 the actual size of the the EYFP (27kda) but both my GST-N-EYFP and GST-C-EYFP appear as the same size on 15% SDS PAGE gel.
Could you suggest something about my problem here..

Thank you


Pages: Previous 1 2