Methods Digest, Vol 46, Issue 16

Suman via methods%40net.bio.net (by sumanknp from gmail.com)
Thu Mar 26 00:56:08 EST 2009


Hi frens,
pleae send me a copy of this method..i think its useful for me
thank you

On Thu, Mar 26, 2009 at 2:05 AM, <methods-request from oat.bio.indiana.edu>wrote:

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> Today's Topics:
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>   1. native PAGE pH (Nicola Traverso)
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> ----------------------------------------------------------------------
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> Message: 1
> Date: Wed, 25 Mar 2009 15:59:19 +0100
> From: Nicola Traverso <ntravers from medicina.unige.it>
> Subject: native PAGE pH
> To: methods from magpie.bio.indiana.edu
> Message-ID: <49CA46C7.70005 from medicina.unige.it>
> Content-Type: text/plain; charset=ISO-8859-15; format=flowed
>
> 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
>
>
>
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-- 
Suman


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