How to do NATIVE PAGE?

Ron Tate rtate at bmb-fs1.biochem.okstate.edu
Tue Dec 10 17:18:56 EST 1996


Thorsten Schmidt wrote:
> 
> Dear Reader!
> 
> I want to separate my target protein from other proteins in low
> concentration
> via native PAGE (poly acrylamide gel electrophoresis).
> What do I have to think about when planning the experiment?
> Where are the differences between native PAGE and SDS-PAGE?
> 
> I heart that one has to calculate the isoelectric point.
> But how can I do this with a 300 aa-protein? (the sequence is known)
> 
> Thank you for your answer!
> 
> Thorsten Schmidt

Thorsten,

During SDS-PAGE you have treated the sample with a reducing agent, a
detergent and boiled it, the result of which is that you have broken
disulfide bonds, disrupted quaterniary, tertiary and secondary structure
and coated the elongated peptide chain with a negativly charged
detergent which associates with all proteins at roughly the same ratio
so that you now have proteins with a negative charge determined by how
big they are.  These proteins then separate out in the gel by size with
the smallest moving fastest.

During Native PAGE you do your best to insure that your proteins retain
thier quaterniary, tertiary and secondary structures.  When the current
is applied the proteins in the sample migrate according to their charge,
their size and their shape.  The protein charge in the particular buffer
system you use is very important as it will migrate toward the pole
opposite of its charge and you want it to migrate through the gel.  So
you either have to know its pI or run two gels, one with one polarity
and the other with the opposite and then be able to determine where the
protein you are interested in is.  You mention that the sequence is
known, there is software available that will calculate the pI of an
amino acid sequence, though I don't know the names right off hand.

Other considerations for preparation involve what you want to do with
your protein after electrophoresis.  These are too many to mention now
but if you have questions and think I can be of help feel free to
contact me by e-mail.

Happy Hunting
-- 
>>>>>>--------------------------->
Ron
Tate                                                                   
Lab of Franklin Leach
Dept. of Biochem. & Molecular Biology
Oklahoma State University 
rtate at bmb-fs1.biochem.okstate.edu
(405) 744-9326
---------------------------------------------



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