SDS Page Problem: Proteins stuck at stacking line - (May/30/2013 )

Hello,

Ive been scouring the forums for help on this and have not found a solution to my problem.

I am producing and refolding a GST fusion protein that has many disulfide bonds that form a dimer of about 110kda when refolded. Reduced is 55kda. I refold in a very specific buffer (ph9.8 Tris, NaCl, KCl, red/ox glutathione, arginine).
Then I purify with a mix of ion exchange and GST beads. I also cleave the GST off of my protein with thrombin.
However I cannot see my protein on the gel, it gets stuck at the top of the gel no matter what I do!
Interestingly enough when I add DTT I see my protein clearly at 23 and 26kda (expexted monomer sizes after thrombin cleavage of GST).
I have tried:
8M urea with and without boiling
Lower concentration with and without boiling
Boiling at 95 vs 70 or none at all
Ethanol precipitation to change buffer to PBS and remove salts.
Dialysis to PBS to remove salts and refold buffer components.

On my WB it shows smears of proteins but only at the stacking line, indicating precipitated protein, but a clear band at the monomer size with DTT.

What else can I do?
My fear is that the protein is refolded but improperly, and as soon as it is unfolded again via sds/heat/urea becomes extremely unstable.
This is further complicated by the fact that the fully refolded protein does not bind to GST beads...I use the beads to capture the GST tag after cleavage. Ion exchange works very well with my protein directly after refolding (ph 9.8).

first of all, never heat your protein in the presence of urea. urea decomposes and carbamylates the protein when heated (even at 37C).

are you saying that you don't use reducing agent when you prepare your sample for sds-page? the reducing agent breaks disulfide bridges and allows for complete denaturing of the protein in the presence of sds. that may be why you are getting good results when you add dtt. you can use dtt or bme in your sds sample buffer.

sds-page will not tell you if your protein refolded properly. if it is an enzyme then you can assay it to determine if the protein is properly functional. x-ray crystallography may tell you if the protein has the proper conformation. usually the protein is assumed to be (mostly) correctly folded if it is soluble after dialyzing away urea.

Hi,

Are you running on gels with different pHs? Maybe the pH change is around the proteins isoelectric point, shifting it to a more positive charge and stopping it from running into the resolving gel.

JoeSus on Sat Jun 1 11:26:55 2013 said: