Loading control for western blot of serum samples...

1D, 2D, HPLC, SDS PAGE, etc. Talk about it here.
Biomarker
Albumin Member
Posts: 88
Joined: Thu Aug 09, 2012 3:28 am

Loading control for western blot of serum samples...

Postby Biomarker » Mon Dec 10, 2012 2:25 am

Hi,

Can anybody please comment on the use of loading control? I am working on glycation PTM (non-enzymatic process). I want to ensure whether i have loaded equal protein on gel or not.

How about the use of commassie staining as loading control.

Biomarker...

Jen_G
Angiotensin Member
Angiotensin Member
Posts: 31
Joined: Mon Jun 27, 2011 12:41 pm

Western of a housekeeping protein is better for a loading control

Postby Jen_G » Wed Feb 20, 2013 10:38 am

Hello
Once a gel is stained with Coomassie you can do a western blot on it. For a rough estimate you can ponceau stain the blot but using another primary antibody for a protein that doesn't change is better. That way you can quantitate the bands to prove that you have equally loaded your lanes. Popular ones are actin, beta-tubulin, or AKT.

MassSpecMan
Carbon Member
Carbon Member
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Joined: Tue Feb 19, 2013 7:08 am

Postby MassSpecMan » Wed Feb 20, 2013 12:23 pm

If you are using the yeast model system, our lab has used PGK1 in the past as a loading control. It is a very good antibody at 1:1000 dilution, and gives very little background.


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