SDS-PAGE migration conditions

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buthercup
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SDS-PAGE migration conditions

Post by buthercup » Thu Mar 04, 2010 9:53 am

Hello every body,

Hope someone could help me.
I'm trying SDS-PAGE for visualisation of polysaccahrides, and I wanted to try different % of polyacrylamide.
Working with protein, I' usually use 12% gels, in a continuous system buffer (Laemmli buffer), and I use 20mA/gel.
To separate polysaccharide, the porotocole I follow (and bibliography) recommend a discontinuous system buffer, and it recommends also to run at constant voltage (so the protein electrophoresis guide I'm using to refresh concepts).
In the very fist paper that evaluate the use of this discontinuous buffer system for polysaccharides, the SDS-PAGE conditions were 16%, 30V while sample were passing through the stacking gel, then 100V when samples entered the separating one.
I've tried this conditions with a 12% gel, and the profile was very nice..., but I'm ready to try a 9% gel, and I hesitate in choosing the voltage...

Can anyone recommend me any voltage condition???

Thanks very much in advance

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JackBean
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Post by JackBean » Thu Mar 04, 2010 11:05 am

IMHO the conditions should not be dependent on gel %.
But should't you have limiting ampers?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

buthercup
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Re: SDS-PAGE migration conditions

Post by buthercup » Thu Mar 04, 2010 12:42 pm

But of course the gel % have an influence...
It's say the equation for the Joule Heat: P (heat) = V * I = V^2/R = I^2 * R

If the % is lower..., then the resistance (R) is lower..., then if I don't want to burn the gel during migration, I have to change (maybe just a little) the migration conditions...

At least this is what I think... :?:

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mith
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Post by mith » Thu Mar 04, 2010 9:18 pm

try it, I don't think the resistance is that dependent on % since the main mode of electron transport is probably the buffer.
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Post by crama » Thu Mar 11, 2010 9:05 am

when you reduce gel % , it migrates much faster than higher %, but there is no chances to burn gel at all. you can choose v constant or amp constant.

rama
Last edited by JackBean on Thu Mar 11, 2010 9:08 am, edited 1 time in total.
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