native PAGE pH

Dear Dr Engelbert Buxbaum

I found this e-adress in bio.net.
I'm a little bit confused about pH in PAGE gels, in particular native
gels. I need your help.

When in SDS-PAGE (discontinuous system) I use
-tris-glycine pH 8.3 as electrophoretic buffer
-tris-HCl pH 6.8 as stacking buffer
-tris-HCl pH 8.8 as resolving buffer
at which pH are proteins separated?
I though at 8.8, i.e. the pH of the resolving buffer, but I've recently
read that they separate at pH 9.5! How can this be true?
Does the pH of the tris-glycine electrophoretic buffer influence the pH
of the resolving gel during the electrophoretic run? if yes, how much?

In native-PAGE (continuous system)
-do I have to use the same pH in sample buffer, electrophoretic buffer
and resolving buffer? Tris-HCl is OK? Must I use tris-glycine in the
electrophoretic buffer? May I change the pH, let us say, between 7 and
10 as I like?

In native-PAGE (discontinuous system)
-how can I set systems at pH different than usual (stacking: 6.8;
resolving 8.8; electrophoretic 8.3)? for exemple, if I desire to resolve
at pH 7.8, how do I have to prepare the other buffers (stacking, sample,
and electrophoresis)?

Thank you in advance

Best regards

Nicola Traverso
Dept Experimental Medicine
Section of General Pathology
University of Genova
Italy

Am 25.03.2009, 10:59 Uhr, schrieb Nicola Traverso
<[Only registered users see links. ]>:



The answer to this question is fairly complicated, and may be found in the
following papers:

@article{Orn-62,
AUTHOR= {L. Ornstein},
TITLE= {Disk electrophoresis: {I}. {B}ackground and theory},
YEAR= {1962},
JOURNAL= {Ann. N. Y. Acad. Sci.},
PAGES= {321-351},
VOLUME= {121},
DOI= {10.1111/j.1749-6632.1964.tb14207.x},
LANGUAGE= {engl}
}

@article{Jov-73a,
AUTHOR= {T.M. Jovin},
TITLE= {Multiphasic Zone Electrophoresis. {I}. {S}teady-State
Moving-Boundary Systems Formed by Different Electrolyte Combinations},
JOURNAL= {Biochemistry},
VOLUME= {12},
YEAR= {1973},
PAGES= {871-879},
LANGUAGE= {engl}
}

@article{Jov-73b,
AUTHOR= {T.M. Jovin},
TITLE= {Multiphasic Zone Electrophoresis. {II}. Design of
Integrated Discontinuous Buffer Systems for Analytical and Preparative
Fractionation},
JOURNAL= {Biochemistry},
VOLUME= {12},
YEAR= {1973},
PAGES= {879-890},
LANGUAGE= {engl}
}

@article{Jov-73c,
AUTHOR= {T.M. Jovin},
TITLE= {Multiphasic Zone Electrophoresis. {III}. {F}urther
Analysis and New Forms of Discontinuous Buffer Systems},
JOURNAL= {Biochemistry},
VOLUME= {12},
YEAR= {1973},
PAGES= {890-898},
LANGUAGE= {engl}
}

@article{Jov-73d,
AUTHOR= {T.M. Jovin},
TITLE= {Multiphasic zone electrophoresis. {IV} {D}esign and
analysis of discontinuous buffer systems with a digital computer},
JOURNAL= {Ann. N.Y. Acad. Sci.},
VOLUME= {209},
YEAR= {1973},
PAGES= {477-496},
LANGUAGE= {engl}
}