How can I reduce background in W blotting with polyclonal serum - from HIS-tagged fusion protein (Aug/16/2005 )
I am after methods to reduce background in western blotting with some serum generated by injecting rabbits with Ni-NTA purified HIS-tagged protein. I am interested in removing HIS antibodies and more general methods of reducing background such as using bacterial lysate and/or pre-immune serum. The protein was purified in BL21 and it will be used to blot human proteins.
just bumping this up as I've had no reply
You could try using an immunoaffinity column containg a His fusion protein or just synthetic His epitope coupled to sepharose.
Just collect the flow through non binding fraction. To eliminate excess antibody loss due to non specific binding to the beads I would add a bit of non-specific protein (ie BSA) as long as you don't mind some of it ending up in your antibody sample.
doing your blot, what do you use for saturation? I used to do it with BSA then I changed with milk and the background reduced! hope it helps!
BTW purifying your antibody should be necessary!