His-tagged protein won't stick to Ni column

klenchin at facstaff.wisc.edu klenchin at facstaff.wisc.edu
Wed Nov 20 16:18:13 EST 1996


In article <3293705D.74E9 at LMB.uni-muenchen.de>,
   Boris Steipe <steipe at LMB.uni-muenchen.de> wrote:
->> 
->> Yeah, and pick up all those proteins that bind to chelated Zn and Cu,
->> but do not bind to Ni (FYI, Ni is much more specific for His, while 
other
->> divalents bind other aromatics as well).
->> 
->> - Dima
->
->Pharmacia and Qiagen material are not the same: Pharmacia sells
->Iminodiacetic acid (IDA) and Quiagen sells Nitrilotriacetic acid (NTA).
->We once checked and found that Zinc would work somewhat better with IDA
->and Nickel somewhat better with NTA. The differences were not dramatic
->though. Check Lindner et al. (1992) Methods 4:41-56.

Sure. Other things being the same, IDA is less specific for His than NTA. 

The difference is not dramatic only in one case - when working 
with well-behaving His-tagged proteins that bind at high imidazole 
concentration (~> 20 mM).

When it comes to purification of non-tagged protein, Ni-NTA is the 
combination that binds lowest proportion of "total protein" form 
crude extracts. 

- Dima



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