Native PAGE stacker pH

Pow Joshi pow.joshi at gmail.com
Wed Aug 16 19:44:26 EST 2006


Can anyone tell me why the Ornstein and Davis Native PAGE recipe for a
>stacking gel is at pH 6.8 when the separating gel and buffers are
>usually around 8.5 and up?  It seems counter-intuitive to drop the pH in
>the stacker... or is this something to do with gel integrity?  I have
>looked through the original Ornstein papers but can't quite work out the
>reasoning.  Anyone know the logic behind it?

For a stacker to work, you want the voltage to drop primarily on it.
> E.g, you want the field intensity, E (V/cm), to be high in the stacker
> and low in the separating gel. The rate of EF is proportional to E.
> That means protein will run fast through stacker and slow down
> once reaching the end of it ==> concentrating action.
>
> All of it requires low conductivity in the stacker. Cl- is a fast ion,
> glycine is a trailing ion. pI of glycine is somewhere close to neutral.
> Which means that close to 50% of glycine molecules in the stacker
> will be neutral and won't conduct current. There you have it: low
> concentration of slowly moving ions = low conductivity = stacking
> action.

..yes, DK is right.... those are the pH conditions that lead to
stacking of proteins, and that's true even for SDS PAGE.... chloride
act as the "leading ion" and glycine as "trailing" ions....except here
due to the SDS binding (1.4g of SDS/1g protein), the proteins acquire
a net negative charge, and their migration therefore depends on the
sieving action of the gel, and therefore the mobility is proportional
to the size...

it's true DK, "biochemistry" seems no longer in fashion....:((
pow


On 8/16/06, DK <dk at no.email.thankstospam.net> wrote:
> In article <44e2be1f$1 at clarion.carno.net.au>, ben.long at yourfinger.anu.edu.au
> wrote:
> >Hi all,
> >
> >Can anyone tell me why the Ornstein and Davis Native PAGE recipe for a
> >stacking gel is at pH 6.8 when the separating gel and buffers are
> >usually around 8.5 and up?  It seems counter-intuitive to drop the pH in
> >the stacker... or is this something to do with gel integrity?  I have
> >looked through the original Ornstein papers but can't quite work out the
> >reasoning.  Anyone know the logic behind it?
>
> Isn't it amazing that the basic theory of electrophoresis is no
> longer taught and in 99.9% cases people using electrophores
> have vague idea why what they are doing works?
>
> For a stacker to work, you want the voltage to drop primarily on it.
> E.g, you want the field intensity, E (V/cm), to be high in the stacker
> and low in the separating gel. The rate of EF is proportional to E.
> That means protein will run fast through stacker and slow down
> once reaching the end of it ==> concentrating action.
>
> All of it requires low conductivity in the stacker. Cl- is a fast ion,
> glycine is a trailing ion. pI of glycine is somewhere close to neutral.
> Which means that close to 50% of glycine molecules in the stacker
> will be neutral and won't conduct current. There you have it: low
> concentration of slowly moving ions = low conductivity = stacking
> action.
>
> DK
>
> _______________________________________________
> Methods mailing list
> t
> http://www.bio.net/biomail/listinfo/methods
>



More information about the Methods mailing list