Loading control for Western blots - (Aug/01/2008 )
I see that many people show a loading control, such as actin, in their Westerns but I am unclear as to how to best approach the task.
Are the blots stripped and reprobed with the control antibody of choice? This seems like a less than ideal option as does cutting the blot.
All of the actin antibodies I've ever worked with are very clean and only give the one band so, unless I previously probed the membrane for a protein at the same size, once I'm done with everything else I simply reprobe for the actin. There really is no need to strip the membrane unless you've got signal from a previous blot in the expected size of the actin. If you already have signal which will interfere with the actin, you must either strip the membrane or if the previous antibody was of a different species, you can destroy the HRP with sodium azide and then reprobe. Cutting a blot is actually an excellent method for getting multiple blots from one gel, as long as the sizes are resolved well enough. Be sure to run marker on both sides to get straight cuts and block after cutting the membrane.