Hello,
I am having problems with beta actin in some of my samples. These are cerebellar samples under denaturing conditions, 10ug protein per well. Transfer looks fine after Ponceau S but beta actin is not detected in some of them. The first 4 lanes are from saline-treated mice, next 6 are PCP-treated. Could anyone help me understand what could have gone wrong?
Thank you,
Lukasz
I assume this is a western blot, yes? Is it possible that PCP could induce expression of b-actiin in the cerebellum? Not sure how this would work, but that is what the data looks like. Is this short exposure? does it come up in the first 4 lanes upon longer exposure?
Hi Gregory,
It is western blot membrane exposed for 1.5 minutes. First 4 lanes are cerebellar samples from saline-injected animals, latter 6 from PCP-injected animals. My saline-treated animals show no beta actin (or very little as in lane 3 of this blot). I am supposed to use beta actin as loading control however I think my control samples have degraded and thus not showing beta actin.
When I probe the membrane for my proteins of interest they stain just fine, but then I cannot analyse the data due to no loading control.
We are looking to do total protein as loading control (stain-free gels) to save these samples and get some meaningful data from them, but if the lower molecular protein like beta actin are missing from the sample, to what extend can we trust other proteins aren't missing too?