Stacking gel recipe for Acidic Native Gel? (View forum version)



ProteinWork

Posted 14 April 2010 - 12:47 PM

I am running acidic native gels for my protein with a pI of around 9.9 to check possible oligomerization. I'm using an acetate-beta-alanine buffer (pH 4.3) to run my gel. I want to include a stacking gel for better resolution. the only stacking gel recipe I found for this system is using 0.25M acetate-KOH (pH 6.8) as the 4x stacking gel buffer. My question is that acetic acid has a pKa of 4.76, so it does not really have much buffer capacity around pH 6.8. Is that a particular reason that the stacking gel's pH is almost neutral?

Also I am thinking of a running buffer of higher pH, e.g., a Histidine-MOPS buffer at pH 6.6. But I could not find a stacking gel recipe for this system. How should I design a stacking gel buffer compatible with the running buffer I choose?

Thanks in advance for any input!

ProteinWork

Posted 16 April 2010 - 08:35 AM

I am running acidic native gels for my protein with a pI of around 9.9 to check possible oligomerization. I'm using an acetate-beta-alanine buffer (pH 4.3) to run my gel. I want to include a stacking gel for better resolution. the only stacking gel recipe I found for this system is using 0.25M acetate-KOH (pH 6.8) as the 4x stacking gel buffer. My question is that acetic acid has a pKa of 4.76, so it does not really have much buffer capacity around pH 6.8. Is that a particular reason that the stacking gel's pH is almost neutral?

Also I am thinking of a running buffer of higher pH, e.g., a Histidine-MOPS buffer at pH 6.6. But I could not find a stacking gel recipe for this system. How should I design a stacking gel buffer compatible with the running buffer I choose?

Thanks in advance for any input!

To answer my own question... I found this paper very helpful:

http://dx.doi.org/10.1016/0003-2697(81)90178-0

Analytical Biochemistry
Volume 118, Issue 1, 15 November 1981, Pages 194-196

A new discontinuous buffer system for the electrophoresis of cationic proteins at near-neutral pH

John M. Thomas and M. E. Hodes

It's a histidine-MOPS buffer system with MOPS-KOH as stacking gel buffer. the resolving gel runs at pH 6.8, so it's near neutral pH. Should be good for monitoring the oligomerization of basic proteins. :D