His tag and second purification step

The Kindly One smccull at jurassic.fisher.calpoly.edu
Wed Apr 23 19:34:21 EST 1997

In article <01bc4e76$93403f60$02cbb2c6 at kaiwan.kaiwan.com>,
>> I have a number of proteins with an N-terminal His-tag, expressed at low 
>> level. Affinity purification on Ni++ enriches them, but it is still only 
>> 10% or so of the total protein. Also, I am convinced that I use enough 
>> Ni++-NTA (Qiagen), but there still is a lot of protein in the 
>> flow-through, so it may be that other proteins aspecifically stick to 
>> the Ni++, preventing my babies to bind.
If you have too many others preventing binding, try diluting sample to 
less than 5 mg/ml!!!! It always works for me when using His-tagged
sampled Ni-NTA.


** Scott D. McCulloch(Bunny Lover)	| If we knew what we were doing,
** smccull at jurassic.fisher.calpoly.edu	| it would not be called     
** BioChemical Effects Research		| research, now would it?
** http://www.calpoly.edu/~smccullo/    |

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