Normalizing protein expression under 24hrs ischemia - normalization with housekeeping genes (May/01/2005 )
I am doing in vitro 24hrs ischemia on cell culture. After that I would like to study some protein expressions. I have tried normalizing the protein level with two housekeeping genes, beta-actin and alpha-tubulin. However, it seems to me that their expression are changed under 24hrs ischemia. So I would like to know if there is any housekeeping gene whose protein level is not changed under such condition. thx..
Check this link www.activemotif.com they have several normalization kits and looking at them might give you good ideas for your experiment. good Luck!
Just thinking that if you are finding that expression is going down could it be that you are seeing significant cell death? 24 hours ischemia is pretty severe, depending on how you are doing the experiment, and therefore will result in significant cell death.
But i did measure the protein concentration before doing western blot.
But i did measure the protein concentration before doing western blot. I use same amount of protein for each sample.
Sorry I cannot think of a suitable protein but ....
If you load the same amount of protein as assessed by a BCA assay or similar I am not sure I would worry too much about normalizing it via western right now. You can always write in your methods or figure legend that you normalized it prior to loading. Otherwise if you think the reviewers will critique you on this one or your differences are subtle so you need to prove there was no loading error, why not check out similar papers and see what protein they used.