6 replies to this topic

[bioxesus]

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.

[mdfenko]

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).

[klinmed]

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

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 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.

[bachai]

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).

[bioxesus]

Thanks, I will try it.

[mdfenko]

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

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 labeled primary antibody made in goats.

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.

[klinmed]

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

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

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 labeled primary antibody made in goats.

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.

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