Hi, it`s not about the protocol, it is about your antigen/antibody combination. In any case you will never get of an antibody COMPLETELY off yor blot, but the amount of antibody remaining depends on the affinity of your specific combination, so protocols working for others may not work for you.

Why do you need to strip your Western blot? Do you want to use different antibodies on the same target (e.g. total vs. phosphorylated) or do you want to detect differents targets on one blot?

Hello, we use a low pH buffer during one hour, then milk for 1h and then we add a new antibody overnight at 4°C. The most important is to alternate mouse and rabbit antibodies. Moreover, antibodies with low signal are easier to strip. For example, we already finish our WB with actin because it's mostly impossible to strip it. 

The short answer is: no, you will always have residual signal from the previous run. Nevertheless this might still allow you to get the answer you want depending on the combination of antibodies/antigenes you are interested in detecting. All comments above seem very useful along these lines and give good suggestions on how to preceed

Thank you all very much.  I have only enough sample for two blots, and I want to maximize the information I can get from those two blots.  I am hoping to probe them for several proteins of similar size.  All primary antibodies are from rabbit, so changing color of secondary doesn't help.  Any suggestions are welcome and appreciated.

Hi Jaclyn,

you could do an IEF in minigel/miniblot format like with EC66452

Novex™ pH 3-7 IEF Protein Gels or others, it is easy.

Thank you, Markus Klotz - I didn't know about IEF, and I'll look into that!