Hello everyone, Can anyone please resolve my problem. I am using NTA resin with some preferential metal for understanding the pulldown of proteins by IMAC technique. I elute my protein by using 100mM EDTA. The problem is that "the amount of protein which I get from elution or from protein through is lesser than the amount which I load onto the column". I don't know how I can elute the protein completely?
Some of the protein may be precipitated on the column. It may only come off by using acid, base, or denaturant washing.
1) you need to make sure that 100 mM EDTA is enough to completely strip your protein from your preferential metal.
2) you need to get data that the binding of your protein with preferential metal does not result in the protein precipitation under conditions used.
3) you need to check that your EDTA stock solution is fresh-prepared and has pH ~ 8.0.
4) depending on the method you use for your protein determination, you need to check if the binding of your protein to this metal or exposure (presence of) to EDTA does not affect the results of your protein determination.
5) you can completely elute your protein if, instead of EDTA you use some acid buffer with pH ~ 4.0, collecting the eluted fraction into a tube with some neutralizing buffer to get pH close to the physiological optimum for your protein function.
Congratulations! This seems to be the first question I've seen on RG regarding material balance for chromatography. The results in the methods you use to strip all protein from the resin will help significantly in determining what is occurring with your protein.
If there is indeed protein product remaining on the resin then there's a reason why it is remaining there while the rest of the product eluted (You might want to employ an orthogonal method for protein determination because there's certainly Ni in your elutions.). Would you actually want to keep the material that doesn't elute well? If not then move forward with the destructive methods of stripping the column. Qiagen has a listing of non- compatible reagents and their concentration (some not so destructive) which you might want to exceed if trying for material balance. You've certainly exceeded the EDTA concentration but combinations of reducing agents, pH extremes, chaotropes, amino acids, detergents, solvents, temperature, proteases may be helpful; you'll have to be able to adjust the protein assay accordingly, perhaps gels.
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