My primary antibody is a poly clonal antibody. Even though I'm using 5% skimmed milk for blocking and 0.1% PBST at three times for five minutes each for washing, I' still getting non-specific signals. Can you give me any suggestions to avoid non specific bindings?
Hello,
Try to block in milk overnight, and wash 4 times 10 minutes.
If it does not work, you may also use 0.15% PBST, but it can reduce the overall signal if your antibodies are not very good.
Hope that helps!
The non specificity may be avoided by washing 6 times for 10 mins each in enough volume of TBS-T (0.1%) and using lower dilution of Primary and secondary antibody, shorter exposure time to the membrane. You have to twik between the different steps you can considerably reduce the non-specificity. Sometimes changing the company of the Antibody also affect this thing!!! You can use the incubation of Antibody in the blocking buffer also!!! All the best!
Hi Angel, I will affect but little bit not too much!!! It also depends upon the affinity of your anitbody towards its antigen, if it is good then does not affect at all if it is low then it affects significantly but still you get clearer picture than the dirty one!
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