Western Blot - Any one ever use serum as a primary antibody? (Jan/13/2009 )
I would like to do some western using different patient serum to look at vaccine response to different antigens that were in a new vaccine. Does any one have an idea about what dilutions of patient sera to use? I don't know where to begin. Thanks.
I have used sera from rabbits for westerns. In this case the rabbits were being used to raise an antibody, so they were being stimulated to have a strong immune response and we got good westerns out with standard sort of dilutions (1:1000ish)
As you are trying to see the response from patients, you will have to titrate each serum against the antigen. However how will you know if this is due to a low response or just a poor antibody sensitivity?
I don't know if it would work, but could you try running the serum on a gel, then add the antigen, allow the antibody to bind the antigen, blot and then probe for the antigen. In theory the amount of antigen bound will be proportional to the amount of antibody response.
I have tried upto 1:50 rabbit serum when checking different sera we were raising. But once we got the rabbit bled finally, we did the affinity purification, and it worked at 1:1000 dilution well.
1:50 gave a whole lot of background, but it still identified specific band. That is the rabbit we chose for further boosters.