Blocking buffer for antibodies generated in goat. - (Oct/08/2009 )

Wich blocking buffer do you use to block the membrane of western blots when a secondary antibody generated in goat? I have used milk or BSA but always the blot apears very black.

Thanks in advange.

i use a mixture of bsa and normal goat serum (2% each) in pbs (no tween) and maintain 2% normal goat serum in the antibody solutions (in pbst).

bioxesus on Oct 8 2009, 02:14 PM said:

I have used 5% skimmed milk with no problems. Have you titrated your secondary Ab? Maybe you are using too high a concentration.

If you still have problems even with an optimal Ab conc you could try a blocking solution compose of fish proteins (I think Pierce Chemical market one).

I would NOT recommend normal goat serum blocking with a SECONDARY reagent made in goat. At the very least the goat immunoglobulin in the serum would consume a large part of your secondary reagent. Blocking with goat serum would be fine with a labelled primary antibody made in goats.

Hope this helps.

5% skimmed milk in PBS + T20 is fine in our lab and other labs I have been to.
Try both titrating the 2nd Ab, and vigorous washing (e.g. 5-6 times, in large volumes).

Thanks, I will try it.

klinmed on Oct 10 2009, 08:32 AM said:

normal goat serum is a blocking agent of choice for secondary antibodies produced in goat. unless the goat in which the secondary is produced exhibits a autoimmune response there should be no binding of the secondary antibody to proteins native to the species in which they are produced.

mdfenko on Oct 14 2009, 05:43 PM said:

Whoops! You are absolutely right..... Sorry for the misinfo.