GADPH Loading Control Western Blot Problem

[jrscientist]

My lab has used GAPDH as a loading control for WB in the past, as it does not change (much...) in response to our manipulation (removal of neural activity). However, I am also looking at differences in these changes over development (mice, age P7 - adult), and GAPDH appears to decrease (in my hands, without a control to compare it to) as the mice age. Does anyone have any suggestions as to a potential loading control to account for both of these issues?

I have toyed with the idea of using two loading controls (one for development, the other for our manipulation), but I hate to have to probe for so many Abs, and I do not seem to get reliable results when I strip the (nitrocellulose) membrane more than once.

Any thought?

Thanks very much in advance.

[admin]

Hello there jrscientist,

not sure why GADPH changes with development. Have you looked at protein concentrations as well or protein synthesis over those times?

Also positive/loading controls do give sometimes funny results. I would try using another loading control such as beta-actin or tubulin and see what that gives.
Good luck