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

[JVM]

Recently we have found in our research that loading controls like GAPDH, b-actin drastically changes in our experiments without affecting the cell viability. Later on when I was looking in the pubmed, it was reported by various people that oxidative stress-induced chemical modification of these proteins and various other treatments can change so called housekeeping genes or protein level in the cells. It is difficult to find an ideal loading control. One can screen few house keeping genes/proteins and adopt one of them which does not change in their specific experiments/ treatments.

[lbmwu]

this may sound like a dumb question... but... i need to use a actin as a loading control but i am not sure how to do this and in what part of the protocol. we have tried GAPDH and this was before me and they said it didn't work.