It would be correct to cut like this only if you are sure that you loaded exactly the same way all your samples. If your target is far enough from a-tubilin, I would load ladders before and after the samples, then cut the membrane according to the molecular weight.

If you are using ECL based detection you can avoid stripping by incubating your membrane with washing solution + 0.1% sodium azide for 30 minutes. This will destroy the HRP enzyme but should keep all the protein still attached. Then you can just probe as normal. This technique is especially useful if you have primary antibodies from different species. Good luck

Dear Christine,

Thanks for your answer.

Most of the researchers have been cutting the membrane according to MW. But here my target and a-tub are very close to each other. As you told, I will obviously load the samples more precisely and vertically cut the membrane. However, I am worried about reviewer comments.

Best regards,

PKJ

Dear James,

Thanks for answering me.

Yes, I am using ECL. Your advise is useful. But I want to save my time actually by avoiding stripping.

Best regards,

PKJ

Can you try PVDF membrane instead of nitrocellulose? With PVDF you can easily remove the antibody and not the proteins.

Do you have access to a fluorescence reader, such as licor or Biorad's chemidoc system? You could run two antibodies at the same time, ie, your protein of interest and then a house keeping gene. That saves a lot of time.

I have also performed fluorescence blots after ECL blots. For instance, I probe with one antibody and develop with ECL. Then I wash the blot with PBS a few times and then add my house keeping gene such as beta tubulin or beta actin and incubate for a few hours. I then add a fluorescent secondary antibody and detect my house keeping gene. Both scenarios work best when your protein of interest and house keeping gene are either from different species or really far apart in terms of molecular weight.