I use biorad marker . Most antibodies m wt around 40/50. I plan cut horizontally to use 3 antibodies per membrane but the m Wt so similar. Can't cut vertically as I have 7 samples. Attached picture .
The caspase shouldn't be too much of a problem as it is quite low. But you could put on one of your 40/50 m wt Abs, develop and image and then just strip the membrane (the abcam website has a protocol for doing this). Then you can add your next ab, develop, and image. It may take a little longer but should work.
The caspase shouldn't be too much of a problem as it is quite low. But you could put on one of your 40/50 m wt Abs, develop and image and then just strip the membrane (the abcam website has a protocol for doing this). Then you can add your next ab, develop, and image. It may take a little longer but should work.
Sally has given excellent advice on what you can do about the two proteins that are very close together. I just wanted to add that this method works best when you are using antibodies raised in two different species. I'm not sure what antibodies you are using but if both the p53 and beta-actin antibodies were raised in the same species, you will have the risk of a band showing up (from previous blot) when you do the second develop even though you stripped the blot.
As for which antibody to use first, there are a couple things to keep in mind. First, you want to use your most sensitive antibody first, this will be the one that is sensitive to stripping. There might not be a way for you to know before you try this out. Often, the less protein you expect to be present the more sensitive to stripping. Also, it's a good idea to start with the protein of interest and follow that with your total protein. Lastly, this isn't really related to this particular blot but is worth mentioning, always start with the phosphorylated protein blot before doing total protein when you are looking at phosphorylation.
You also want to be careful to not burn your blots when you develop. Even with stripping, the burned regions (over developing) will appear as cold spots (white lines) when you do the second develop. Good luck!