Big problem in GST Protein purification - (Jan/10/2008 )
Hi everybody 
Recently I was success in joining 2 genes and cloning them as single gene in pGEX-4T1 vector for expression. Although this protein was expressed in soluble form but its amount was quite small. And I already sequenced its and know that its sequencing is Okay ( GST-Gene), besides I check by Western Blot using GST anti as 1st antibody, and the result means that my protein already bind with GST. 
But problem is when I purify it using Glutathionine 4B sepharose column. My protein can’t bind with resin and nothing in elution fractions. I already check purified ability of this column by run protein from pGEX-4T1 empty vector, and everything is okay. 
Everybody, have you ever met this condition like me? Please recommend to me how to solve this problem.
Thank you very much
Luyen
Recently I was success in joining 2 genes and cloning them as single gene in pGEX-4T1 vector for expression. Although this protein was expressed in soluble form but its amount was quite small. And I already sequenced its and know that its sequencing is Okay ( GST-Gene), besides I check by Western Blot using GST anti as 1st antibody, and the result means that my protein already bind with GST.
But problem is when I purify it using Glutathionine 4B sepharose column. My protein can’t bind with resin and nothing in elution fractions. I already check purified ability of this column by run protein from pGEX-4T1 empty vector, and everything is okay.
Everybody, have you ever met this condition like me? Please recommend to me how to solve this problem.
Thank you very much
Luyen
Protein purification can be quite tricky. Have you tried purifying another GST-fused protein? This is a good way to check if your buffers, column, technique, etc. is working. If this purifies okay, then you can move on to your protein. What kind of elution buffer are you using? You could try to increase the concentration of the competitive inhibitor to try to force your protein off the column. Are you seeing your protein in the flow through or wash buffers? If so, this means it is not binding. Try to narrow down where the problem is, and it will be much less overwhelming.
I am willing to bet that your expressed fusion gene(s) are interfering with the GST tag and it's ability to bind glutithione. You can detect the presence of the GST tag in a western because it denatures the protein but you are trying to purify the folded, native conformation protein. Since you were able to purify the GST alone you know that the buffers, column, ect. are fine. The problem is clearly specific to this protein. You may need to put the GST tag on the other terminus of the protein or try a different tag altogether. Looks like you may just be the victim of scientific bad luck.
Hi,
I'm sure you've thought of this but when purifying GST-fusions on a column the flow rate is very important. You should apply the protein at no faster than 0.2ml per minute as the interaction kinetics between GST and glutathione is quite slow. Also, do you have any denaturants in your protein prep (Triton-X, NP-40 etc) as these may be causing your protein to unfold - dialyse into PBS before loading the column. If all else fails you could try a batch purification in which you simply incubate your lysate with a glutathione slurry with mixing (this could be left to bind overnight at 4 degrees),
Hope this helps,
P
Batch purification of GST-tagged proteins is a good method worthing trying. It is quick, efficient and cost-saving.
Generally it is sufficient to incubate total cell lysate with GST agarose for 2 hr.
I'm sure you've thought of this but when purifying GST-fusions on a column the flow rate is very important. You should apply the protein at no faster than 0.2ml per minute as the interaction kinetics between GST and glutathione is quite slow. Also, do you have any denaturants in your protein prep (Triton-X, NP-40 etc) as these may be causing your protein to unfold - dialyse into PBS before loading the column. If all else fails you could try a batch purification in which you simply incubate your lysate with a glutathione slurry with mixing (this could be left to bind overnight at 4 degrees),
Hope this helps,
P
Generally it is sufficient to incubate total cell lysate with GST agarose for 2 hr.
I'm sure you've thought of this but when purifying GST-fusions on a column the flow rate is very important. You should apply the protein at no faster than 0.2ml per minute as the interaction kinetics between GST and glutathione is quite slow. Also, do you have any denaturants in your protein prep (Triton-X, NP-40 etc) as these may be causing your protein to unfold - dialyse into PBS before loading the column. If all else fails you could try a batch purification in which you simply incubate your lysate with a glutathione slurry with mixing (this could be left to bind overnight at 4 degrees),
Hope this helps,
P
Hi, I have the same problem, as my protein isn't binding to my column... I've tried o/n binding at 4C to no avail.
Did you mean trying binding 2hr at room temperature?
Andy
Did you mean trying binding 2hr at room temperature?
Andy
[/quote]
HI
i tried binding my protein for 2 hours at room temperature and i got very good results.
Even i have a similar problem, will increasing distance between GST tag and protein my interest improve binding of fusion protein on GST column as interference in folding with the protein of insert might become less . ex. using PGEX 6P-1 or even 2T-K instead of 4T-2 
Ok, this might work, it might not. All care, a bit of guesswork, no responsibility.
I would try changing the pH of your protein solution. It could be that the different pH will disrupt the interaction between your protein and GST. That way, you should be able to recover the GST binding to the column. I would suggest you need to cross over the pI of your protein, in order to change the ionic environment.
A word of warning: you need to be careful about changing the pH across your protein's pI. The change needs to be fairly rapid, or you increase the risk of aggregation. Test with a small amount of protein first. As with all ionic interactions, you want to be at least 1 pH unit clear of the pI.
All the best...
Recently I was success in joining 2 genes and cloning them as single gene in pGEX-4T1 vector for expression. Although this protein was expressed in soluble form but its amount was quite small. And I already sequenced its and know that its sequencing is Okay ( GST-Gene), besides I check by Western Blot using GST anti as 1st antibody, and the result means that my protein already bind with GST.
But problem is when I purify it using Glutathionine 4B sepharose column. My protein can’t bind with resin and nothing in elution fractions. I already check purified ability of this column by run protein from pGEX-4T1 empty vector, and everything is okay.
Everybody, have you ever met this condition like me? Please recommend to me how to solve this problem.
Thank you very much
Luyen
Hi..
im too facing the same problem, using vector pGEX 4T-3 and cloned it with protein, protein is being expressed bt on binding it to glutathione sepharose ,im not getting anythng....protein is not getting bound to the columm, im doin the whole experiment at room temp..shud i switch oevr to 4 degree celcius, using 1mM IPTG to express protein..freezing the pellet at -80 thn resuspending the pellet with PBS buffer, adding 1mM DTT 1mM EDTA, .25% sarkosyl( to solubilise protein from inclusion bodies),
then sonicatin for 10 secs, 4 times at the speed of 2 to 3.
adding in triton X 100 and thenadding glutathione slurry, but gettin nthng on gel.protein is nt binding...pls help me!i all jinxed rite nw!