Amount of a polyhistidine tagged protein in an elution buffer having 30mM imidazole just after an elution with a 350 mM imidazole containing elution buffer is significantly more.Can anyone please give an explanation for this?
You mean you eluted first with 350 mM imidazol and then with 30 mM imidazole and you got more protein in the second fraction? That doesn't make much sense, why would you even do that? :)
Are you sure you had long enough elution with the first fraction to be sure that nothing of that is in the second fraction?
How did you determine the amount of your protein? Could it be that imidazole interferes with that determination?
Of course, if you mean that you eluted first with 30 mM imidazole and then with 350 mM imidazole and you have more protein in your first fraction, that's fine. Often the majority of the protein elutes with lower concentrations of imidazole. However, the purity tends to be higher in fractions with more imidazole (but doesn't have to be).
I FORGOT TO MENTION, THIS CASE IS ONLY FOR SOME PROTEINS WHICH ARE BINDING TO BEADS WELL,BUT NOT COMING IN ELUTION BUFFER.
For checking remaining protein amount in beads after elution ,i used to make a slurry of bead in a buffer of 30mM imidazole ,but accidently i found even if i am increasing the amount of this buffer and loading very less in a SDS PAGE,I am getting good amount of protein than the normal elution buffer having 350mM imidazole. So intentionally I tried to elute the protein in lower concentration of imidazole just after a higher imidazole elution and I succeed, but couldn't get any proper explanation.
The elution off IMAC with high imidazole is not instantaneous, it usually requires some equilibration in the high imidazole buffer. What is the volume of the column and of the elution beffer? Do yuo wash with low level imidazole prior to elution?
I does not make much sense that protein is able to bing IMAC resin in 0.3 M imidazole, but not ten fold lower. Surely if you just try elution with 30mM imidazole WITHOUT the 0.3M imidazole first, nothing would elute, correct?
Due to degradation issues(for conserving time), I won't use any column for the elution.What i am following is after washing ,beads bound with proteins are resuspended in elution buffer(350mM imidazole),centrifuge and protein containing supernatant will be collected.
For 100ml of culture ,I use 100 microltr of elution buffer to elute.
Actually, imidazole is a quite potent chaotrope and at high concentrations may have adverse effects on several proteins. Is it possible that your protein partially unfolds at 350 mM imidazole and aggregates on the resin, which prevents its elution? Then, when you lower imidazole concentration to 30 mM, protein folds back to its normal form and concentration of imidazole is still enough to elute the protein out.
If you have functional assay for you protein, you can get activity (or binding) measured with/without 350 mM imidazole.
@Jijumon - did you try the elution with 30 mM imidazole first? Just after the wash?
Jijumon - it seems to me you have some "problem" with the common His-tag affinity chromatography. Have you ever tried to follow the "classical" purification steps? Have you tested whether your His-tagged protein had really been bound to the resin? Have you checked that after binding the His-tagged proteins there was no leaking off from the column? Have you determined the minimal imidazole concentration for eluting your bound protein from the affinity resin? You have described a "batch protocol" which seems to be very "unusual" for me. I would recommend to do step-by-step the above mentioned protocol to be able to see clearly where the problem comes out.
@Tomáš Hluska ..i tried elution with direct 30 mM Imidazole first ,I didn't get any protein.
@Alajos Bérczi .
#What I am following is not a chromatography, I 'm using Ni-NTA Agarose beads.After washing ,beads bound with proteins are resuspended in elution buffer(350mM imidazole),centrifuge and protein containing supernatant will be collected.
# I didn't tried any classical purification steps for this protein .
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