Help on using different antibodies in the same membrane - (Nov/25/2011 )

Hi everyone!

I am performing some Western Blottings against different target proteins (MCM7 - also known as CDC47; beta-Actin; 3beta-HSD...) in nitrocellulose membrane. In order to use the same membrane, I am using a stripping protocol to wash the antibodies in every Western Blot "cycle".

My main concern is how many times I can strip the same membrane to investigate different proteins. I also would like to know if I can strip the membrane, wash in buffer, dry the membrane and freeze it to use it later. If it is possible, how should I start the protocol after thawing the membrane?

Thanks for your help!

You can try cutting your membrane after the blocking step if you know the sizes of your proteins of interest. b-actin = 40kDa. If the sizes are too close together 40,45,50 kDa, then it's harder to cut your membrane; if it's further apart: 20, 50, 100 kDa, then you can cut it to give you 3 strips which you can stain with different antibodies.

Thanks for your reply!

Unfortunatelly I can not cut the membrane, as the sizes of the proteins are, in some cases, close. That's why I use a stripping protocol. Indeed, I am having nice bands which are very specific, so the stripping protocol seems to be working fine.

The problem is that I would like to keep using the same membrane to test several different antibodies (I don't know how many times I can strip the same membrane). In addition, I would like to and test if I can freeze a membrane that I am using and start Western again. Have you ever tried this before?

I have an idea.. if they are close, try having antibodies raised in different species. So detect one antibody from rabbit and develop the blot first, then re-incubate with second primary antibody raised in different species and then develop the blot. I never tried this but I've been wondering whether it would work.

zienpiggie on Sat Nov 26 07:38:57 2011 said:

Problem with this is that the more antibodies you put on the same membrane at the same time, the more background you end up having no matter how long you block it. Because you end up needing to add multiple secondaries. And its rare to find other species other than Ms, Rb and maybe Gt.

agodoido on Fri Nov 25 15:03:27 2011 said:

Haven't tried stripping nitrocellulose but it's certainly possible with PVDF. Only issue is that it takes longer to get your final results. If your bands are not super close, you can try to restain your membrane with another antibody while retaining your previous bands. You could wash off ECL, re-block it, then reprobe.

Hello agodoido

I had done stripping of PVDF membrane,it works well.i tried 2times.it was fine. but stripping protocol should be standardised. once i kept in stripping solution 30 mins for instead of 15 mins, then i lossed protein.all the best for yr wb.tell us your experience.

If you plan to strip and use the same blot for many times for different proteins then load maximum protein you load. In that case you can strip 3 or 4 times.

Some time i use same membrane for two different proteins (only if your antibody is so specific to get single band ) or size of protein differ at-least 20 to 30 kD.

Different host secondary antibody also works.

Also i suggest to use gradient gel for detection of multiple protein in same blot

I personally try to avoid stripping whenever possible by the techniques mentioned by the others. If your primary antibodies are from different species, then you can probe for anti-rabbit, then kill the HRP enzyme with sodium azide (0.05%), and then probe for anti-mouse. If your antibodies give very different signal intensities, start with the most faint signal, expose your blot, then reprobe for the one that gives the next strongest signal, and repeat until you have done them all. If all of your antibodies strip off well, and the other techniques don't work, then there really is nothing wrong with stripping.