nondenaturating PAGE

Jeremy M. Murray bmbjmm at bmb.leeds.ac.uk
Mon Mar 23 11:05:57 EST 1998


Silke Beismann wrote:

> Who can give me some hints?
> I want to carry out a nondenaturating PAGE. Neither me nor anyone else
> in my lab has ever done this before.
> What is the best buffer system? Can I cast the gel in the same slab gel
> casting device used for SDS-gels?
> What picture would one expect to find if I load the gel with two
> proteins I expect to form a complex? One is 23 kDa with a pI of 6.79 and
> a net charge of -2, the other one is 28 kDa with a pI of 5.06 and a net
> charge of -8.
> I will be happy about any help I can get!
> Silke

 Yo Silke
its real easy
obviously u have to ensure all your equipment is nice and clean.
but apart from this, just leave out the SDS and the 2-Mercapt. (and don't
boil)
i have run many native PAGE gels using this protocol
stacking gel pH6, resolving gel close to pH9 (as in the BioRad protocols)
i use DNA loading buffer to make my proteins sink
and have always been able to resolve my protein (which has a pI of 6)
however, proteins R no longer separated on the basis of Mr,
charge is the now tthe most important factor.
but because your protein have a quite low pI this should not be a problem.

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Jeremy Murray                            MAIL: bmbjmm at bmb.leeds.ac.uk
Biochemistry & Molecular Biology         TEL:  +44 113 233 2591
Leeds University, LEEDS LS2 9JT          FAX:  +44 113 233 3167
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