Hello, I purified a protein using Dynabeats His tag protocol, the pH of the binding/wash and elution buffers were 8. The suspected His tag protein was seen but with presence of one more band.
I have experienced the problem. My washing buffer at 100 mM imidazole pH7.5 and my elution buffer at 500mM imidazole pH7.5. I got 2 bands, one is my targeted protein, the other is a smaller size band. Even after purifying with ion-exchange chromatography (FPLC), the smaller size band was not removed. I expressed my protein with BL21(DE3).
The two most likely possibilities that come to mind are that the second band is a degradation product (which still has the tag) or that it is simply a contaminating protein. There are a number of proteins in BL21(DE3) that are known to bind non-specifically to His affinity resins; you might want to try NEB's NiCo(DE3) (sp?) which is a multiple mutant devoid of the most common contaminants found in IMAC columns.
I tried the superdex column and sometimes it is good.
Some times the peak is not clear and I have to waste the unclear part, so purity or yeild, you may have to choose one.
Good luck!
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