Ponceau S as loading control on Westerns? - (Jun/01/2006 )
My Boss wants me to do my western validation (loading control) with ponceau S!
I want to do quantitation of protein amount with about ten different samples always loading the same protein amount as determined by bradford assay.
In my opinion ponceau s isn't sensitive enough to see smaller pipetting or bradford errors so I wanted to use a "housekeeping protein" like beta-actin or tubulin...
Does anybody know the prevailing opinion to this subject?
Is there any literature I can refer to?
well that's not litterature based, but i think that ponceau is the first check just after transfert, for you to lknow if you have to set up an other gel or not
But i have experienced gels that are equivalent regarding ponceau loading, and my loading control blotting showed that the quantities were not equivalent.
moreover, i've never seen a loading control by ponceau in publications.
Some reviewers insist on a house keeping protein as control for equal protein level. So u might need this control to publish.
Do both. I use SYPRO Ruby Blot Stain to check transfer efficiency and uniformity. This is a fluorescent stain though so you need a laser to see it. I then probe for beta-actin as well to check for differences in actual protein levels.