Antibody reuse for Western blot - (Aug/19/2008 )

We are having some serious problems with western blots.
After testing different conditions and reagents we think that the problem might be due to the reutilization of the antibodies.
Does anyone has already had this kind of problem? Is the antibodies reutilization recommended?
Thanks a lot for any help.

Sadly, i would say it depends on the antibody you are using. We routinely reuse some of our antibodies, but some others we use are only good for one western.

What kind of problems do you have?

We cannot detect anything on the film. First we thought that the developing reagents were not working but after changing that we realized that when we used a fresh aliquot of the secondary antibodies it worked better. We normally reuse both primary and secondary antibodies.

we normally reuse primary antibodies three times. However, we always use fresh secondary antibodies

Most primary antibodies can be used a few times, especially is you add 0.05% sodium azide to stop bacterial growth. However some antibodies don't seem to like the azide so I have some dilutions that I filter and freeze after every use. Then again, there are some antibodies that just don't work twice and have to made fresh every time. In my experience this has been more for IF than westerns though. I never save or reuse secondary antibodies.

Agree, we never reuse secondary antibodies. I guess there's your problem ^^

we normally reuse primary antibodies three times. However, we always use fresh secondary antibodies

I think you have to use fresh second antibody because of the enzymes (HRP or AP), probably they won't work that well when you thaw, use and freeze them for once and then again. The enzyme function is essential for your detection. Do not save money on this point, you will only get poor results. To reuse the first antibody is possible, if it is a really good one giving fat signals. Otherwise it is better to use fresh one here also.