Connoisseurs of Ni NTA

Alex Alexandre.Blais at crchulNOSPAM.ulaval.ca
Mon May 14 15:04:23 EST 2001


Hi Michael

You are probably experiencing disulphide bridge formation during the
stacking phase of your SDS-PAGE (I assume that you detected the different MW
forms by western blot following SDS-PAGE). It has nothing to do with the
NiNTA purification by itself, but the fact that the protein you are looking
at is (probably) highly pure means that it mainly forms inter-molecular S-S
bridges with itself (explaining the multiples of 22 kD)

Have a look at :

Crow MK, Karasavvas N, Sarris AH.
Protein aggregation mediated by cysteine oxidation during the stacking phase
of discontinuous buffer SDS-PAGE.
                       Biotechniques. 2001 Feb;30(2):311-6.

This paper gives you an explanation to the problem and two ways to avoid it
(using iodoacetamide to block reduced -SH or using agarose stacking gels)

Good luck

Alex



Michael Witty wrote:

> Here is one for connoisseurs of Ni-NTA.  When I loaded a protein (22kDa)
> onto Ni-NTA, I eluted, roughly, 22, 44, 66kDa.  This protein has
> disulphide bonds and I suspect some kind of bad breaking of disulphide
> bonds and reforming between molecules.  Does anyone have similar
> experience of this kind of thing?  Mike.




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