GST fusion protein won't elute

Curt Ashendel ashendel at
Mon Nov 14 22:29:25 EST 1994

On 13 Nov 1994 00:19:36 GMT, 
T. S. Pillay  <tpillay at> wrote:

>My colleague working alongside me has this problem:
>He has tried to purify the GRB2 SH2 domain but he finds that the protein
>does not elute off the beads with glutathione(GSH).  I know this sounds
>weird but has anyone experienced a similar problem with this or any other
>fusion protein.  He gets good induction of expression when looking at
>whole cell lysates but very poor yields with purification.  When he 
>takes the GSH-sepharose beads after elution with GSH and boils it in
>laemlli buffer- he finds "tons" of the fusion protein indicating that the
>fusion protein is sticking avidly to the beads and not eluting off.
> Any ideas/suggestions would be gladly appreciated.
These aobservations go against chromatography theory, so one of your 
friend's assumptions must be incorrect.  Some possibilities to check:

1.  Is he eluting with REDUCED glutathione (not oxidized?).

2.  Is the pH of the elution buffer corrct?  If not buffered strongly 
enough the GSH (a weak acid) reduces the pH. This actually elutes GST, but 
if it is too low, it may denature it on the column.  If you are not sure, 
check it directly.

3.  Is he eluting with a high enough concentration of GSH?   
5 mM has worked for me but I usually use 10 mM.  

4.  Is the extract that is being loaded soluble to the point of being 
non-turbid?  If not the protein may be trapped in aggregates.

5.  Has he tried a positive control,  such as empty vector (plain GST)?  
This will tell him if it is procedural vs intrinsic to the protein being 
expressed.  BTW, this particular protein (GST:Grb2-SH2) should express 
beautifully as soluble material. 

I wish you friend luck in solving his problem.

Curt Ashendel
Purdue University
West Lafayette, IN
ashendel at

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