Partial Purification; no activity

[galata44]

I am getting partial purification of my protein via Ni-His tag affinity chromatography but when I perform activity assays it turns out there is no activity. I have a separate pure protein (of the same class which should use the same substrate) I used as positive control so there is no problem with the assay reagents/technique. Any suggestions on why I may be seeing no activity here, can any co-purified products lead to inactivity?

Thank you
MG

[mdfenko]

in what buffer is the protein? the positive control?

do you use urea in the elution buffer? if so, do you refold the eluted protein?

are you sure the his-tag doesn't interfere with the conformation of the protein?

[CrowSan]

As Mdfenko suggests the protein may be denatured or incorrectly folded following purification. You may also need some metal ions in there for full activity (e.g. many proteins co-ordinate around zinc etc).

[relaxin]

I agree with the two earlier posts, your protein is probably not in the native conformation, and hence no biological activity. There is also a chance that the high level of imidazole used to elute the protein off the affinity column may inhibit the activity of your protein. You can test this possibility by adding some of your eluted protein to your positive control.

[galata44]

Hi,

Thank you for your replies. My protein is being His(6X) tag purified, with no urea. The buffer is TRIS-Cl (pH7.2) and the activity of the positive control is not affected by the high imidazole...... interestingly though (and frustratingly) there appears to be product corresponding to activity of the positive in the assay for my test protein, although they are run in separate assay tubes; and I have tested all the buffer, cofactor (Mg, and Mn) reagents for contamination with this product but there does not appear to be any contamination. The positive is in the same class of proteins as the test and they both accept the same substrate. Is it possible that two proteins in the same class will react differently to differing levels of imidazole, shall I reduce the amount of imidazole in the elution buffer for the test protein?

MG

[mdfenko]

you can try reducing the imidazole concentration in your eluted protein to see if that is causing your loss of activity.

have you assayed for your protein prior to application to the ni-column? the his-tag may be influencing the conformation of the protein. you may have to put the his-tag on the other terminal.

[relaxin]

You can remove the imidazole from your purified protein by dialysis.