nondenaturating PAGE

Li Feng fengli at aecom.yu.edu
Thu Mar 19 16:43:20 EST 1998


I think there is something need to be classified here.  Beside the
generally popular denatured SDS-PAGE, I would say there are two other
variations:

1. Non-denatured SDS-PAGE.  In this system you don't add
beta-mecaptoethanol in your sample buffer and don't heat the sample.  But
you need SDS to negatively charge the proteins and SDS can be used in all
the buffers.  The result is that the potential S-S bonds in your protein
are not broken, but the protein will still migrate according to the its
size (or the SDS charges on its surface).  It is used to check if your
protein is a dimer or oligomer.

2. Native PAGE.  Here you don't add SDS in any buffer, nor do you add
reducing reagents or heating the sample.  In this case, the migration of
your protein on the gel will be determined by its own charge in the buffer
as well as its size.  The normal protein standards will not work in terms
of MW determination.  This method can be used to identified some unknown
or recombinant proteins.

It's all depend on which goal you want to achieve.  Most of the time,
people just want to see if their protein is a dimer.  Then just run
non-denatured SDS-PAGE, and you can still evaluate the MW of the protein
by normal standards.


In article <351151B0.535A at biochem.uni-kiel.de>,
usommer at biochem.uni-kiel.de wrote:

>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
>
>Hi Silke,
>try your normal PAGE conditions without adding SDS in any buffer you
>use. Maybe you have to vary the concentration of the resolving gel.
>Normally, the complex should be slower, but find it out yourself.
>And perhaps you may find something in a protein purification book in
>your library?
>I hope that helps.
>        Ulf

Li Feng
Liver Center
AECOM



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