We alway use several time for western blotting. Actually, at least with some antisera, the background gets better after a few uses, so sometimes we did "mock" blots with new dilutions before using the antibody dilution for an important blot e.g. for a publication. Since we had a fairly high thoughput, we never froze the dilutions, but kept them at 4°, but added some azide to prevent microbial growth.

Hi,

I was reusing milk-diluted antibodies and generally they work. One of my friends was using it about 15 times, every time freezing and defreezing it.

Normally I stored it in -20C. Do not keep it in 4C, because you can have bacteria or fungi in it.

You should at least try, they should work.

Dear,

If your antibody is not very diluted it should work and you definitely need to try. But remember that after the use you should store in (-20 or -80) and not at 4C.

We alway use several time for western blotting. Actually, at least with some antisera, the background gets better after a few uses, so sometimes we did "mock" blots with new dilutions before using the antibody dilution for an important blot e.g. for a publication. Since we had a fairly high thoughput, we never froze the dilutions, but kept them at 4°, but added some azide to prevent microbial growth.

it's not advisable. Normally you are adjusting your antibody concentration to an optimum (normally 0.1 µg/ml ... 1 µg/mL), optimized for concentration and incubation time . You are doing this to avoid to waste to much of the antibody. Due to the incubation you will lose always some antibody. If you would like to know if it's work, just do a kind of experimental set up for an lower detection limit (serial dilution of your antibody). If you are facing differences between this concentration, than you should avoid the reuse. If you didn't see differences than you have already to much antibody in your incubation buffer.

The better way to get comparable results in western blotting is to optimize the conditions and avoid by this way the waste of antibodies.

I always stored such antibodies for western blotting at 4 °C as I used it really a lot. As the others said, in case you do not use it very regularly, you should store it at -20 °C. As each antibody can react differently to freezing, first try if your antibody still works after freezing it before you freeze large amounts.

Susanne - how long could you store your antibodies at 4C? did it have milk in it? I have my antibodies in milk for 2 months at 4C and I want to know if it's ok to be used?

Annemarie Honegger- how much NaAzide do you typically add in your primary Ab solution?

what is the role of milk in this process?

1) blocking

2)non-reacting with the antibodies

3)cheaper source  

what are the other reasons?