Native PAGE @ pH < 6.5

Achim Recktenwald achim at ibex.ca
Mon Mar 11 09:48:49 EST 1996


Christopher Walentas (Bioc) wrote:
> 
> I was wondering if anyone had a buffering system that they could
> recommend for the resolution of two proteins using native PAGE at
> a pH at or below 6.5?  I've got a mixture of two species which
> differ by a series of histidines, and need to utilise their charge
> differences (which are only relevant below their pKa), but cannot
> go too acidic due their pI is around 4.  I've tried citric acid
> at pH 5.5 (100mM) but with not much luck-- the proteins are 100kDa
> and hardly made it into the resolving gel by the time the BPBlue
> had scooted off the bottom.  I've looked into a number of Practical
> Approach-type reference manuals, but for the most part they are too
> general and of little or no help.  Any advice in this matter would
> be most appreciated.
> 
> Cheers,
> 
> tripp


I just found in a copy of an old thesis the following recipe:

Electrodebuffer: acetic acid/beta-alanine pH 4.5
   a 10x solution: 312g beta-alanine + 80mL acetic acid, 
                  add water to 1L

Separation gel:   acetic acid/KOH pH 4.3
                  26.93g KOH + 172mL acetic acid,
                  add water to 1L

Stacking gel:      acetic acid/KOH pH 6.8
                   26.93g KOH + 29mL acetic acid.
                   add water to 1L

I never tried it myself, so don't sue me, if it doesn't work.


Achim

_______________________
Achim Recktenwald
IBEX Technologies, Inc
5485 rue Pare
Montreal, PQ, H4P 1P7
Canada



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