Detection after his-tag purification (was: Eluting his-tag proteins without imidazol)

Holger Neumann holger.neumann at ruhr-uni-bochum.de
Sun Apr 25 05:34:49 EST 1999


Hi netters,

I have a different question on purifying proteins with Ni-resins.
I tried to purify multiple mutants of a kinase.  After batch purification
(elution with imidazole) I performed dialysis to change buffer.  Activity
assays using [32-P]-ATP worked well.
PROBLEM:  Detection after purification failed !!
I tried western blotting detection using different antibodies, silver
staining, Bradford's colorimetric method and even all this methods after
concentrating protein samples with different methods.  Nothing worked, I got
no result.
Please help !!!   Where's the problem ???

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                                               Holger Neumann
                                                 M.D. student
                                          University of Bochum
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