which internal control for my western blot? - (Mar/09/2008 )
I'm doing a quantitative western for a protein with 56 kD molecular weight. I'm wondering if I can use actin as the internal control? wouldn't the actin band cover the band representing my protein of interst? I have to use a polyclonal antibody for my protein and it is also crossreacting with some other proteins in my sample which makes me more concerned about my internal control overlaping some of these bands.
How about GAPDH? It's about 36 kD, this wouldn't interfere with your protein of interest.
then what is the protocol for probing control? should we first probe the protein of interest, then wash off the detection reagent and then probe the control ? should the secondary antibody for protein of interest be different from one for control protein?
Yes, that is the way it's best. Especially if your first protein is in low amounts or hard to detect. I strip after my first protein detection and probe against GAPDH, there is a great antibody from santa cruz which is labelled with HRP so you don't need a second antibody.
Thanks Biomaus, and do you think that would detect an insect protein?
You mean this GAPDH antibody? In the datasheet detection of insects is not explicitly mentioned, I think it's kind of trial and error. It is only tested for mouse, rat and human. The cat-nr. is sc-20357 if you would like to try it.
But if you have an Actin antibody which is good working and known to work for insects, you could also try the routine with this antibody. First detect your protein, than strip and reprobe with Actin. Reactions from the first round should not interefere after stripping.
beta-actin is 42-44 kDa and will not interfer with 56 kDa protein if separated properly