Western Blotting : Can longer incubations require more washing? - (Jun/20/2008 )
I have a question about a recent problem I've had with my Western blots. I am trying a new batch of antibody (an antibody that has worked very well for us in the past, and is commonly used in published studies) and have not been able to get good signal. I think both times I have done the incubations with the primary antibodies they have been done for two days at 4C. I usually cut my membranes in half prior to blocking/antibody incubation so I can probe for my protein of interest as well as a loading control, without going through the membrane stripping process. When I developed these blots, I was able to get pretty good signal from the loading control, but no signal from my protein of interest unless I did a long exposure - then I have too much background for the data to be of any use. I was wondering if I might need to increase the wash times if I do a long incubation like this? Maybe I am getting too much blocking of the band of interest with such a long incubation time?? At first, I thought the antibody batch may have been bad, but this would be our second bad batch in a row from Abcam, which I find to be somewhat unlikely (I think it's more likely something I am doing wrong).
what is exposed long? first or second Ab? anyway, I do not think that the normal washing program, which washes unbound Ab away, should be pro-longed. However, you may test various washing programs to find optimum conditions
I think overnight incubation approx 12 hours or something more than 8 hrs is ok.
I recall that after 24 hrs of incubation i needed more exposure time. Of course it depends on many parameters but usually there is no need to incubate for more than 12 hrs.