Protein SDS-PAGE

[Richard P. Grant]

In article <73fn8p$4uf at bellboy.ucc.uconn.edu>, "Qiang Zhang"
<qiz97001 at uconnvm.uconn.edu> wrote:

> hi, everyone, i have a question about Protein SDS-PAGE. i run the gel which
> is 0.75mm thickness, 2cm height of stacking get plus 12cm height of
> separating gel under constant current of 30mA for two gels(15mA for each).
> it runs so slowly that takes about more than 10 hours to run to the bottom.
> the time is much longer than that in my lab's protocol. the concentration of
> the stacking gel and separating gel are 3% and 10% respectively. i am sure
> that there is no error in my all buffer. is there anyone who has experience
> in it can give me some advice about why it is so slow and what i should
> check about it. how long do you spend to run the gel like this? thank you
> for your help and advice.

Hi, 

it sounds to me like it *is* an error in the buffer.  At a guess, you have
resolving gel pH in the stacking gel.  I'd get some pH paper and double
check your 5x buffers. 

Stacking gel should be 0.125 M Tris-Cl pH 6.8 (final)
Resolving gel should be 0.375 M Tris-Cl pH 8.8 (final).
The running buffer should be pH8.3, but if you make up @192 mM glycine, 25
mM Tris base (0.1% SDS) then you don't adjust the pH.

If you're unsure, throw them out and start over.

I've got things mixed up dozens of times . . . and that's just by picking
up the wrong 5x stock!

-- 
Richard P. Grant MA DPhil     |             rgrant at cmtech.co.uk
Senior R&D Scientist          |             work: www.cmtech.co.uk
Cambridge Molecular           |      home: www.avnet.co.uk/adastra           
           -- +44 1223 508345 - the Number of the Yeast --