secondary antibody - (Apr/12/2007 )

Does secondary antibodies give bands in western blots without primary antibody? If so, how to avoid that?

What if our interest of protein falls in the same range which secondary antibody alone also reacts?

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q1: yes
a1: appropriate dilution of 2nd Ab; in most cases, a higher titer than 1:1000 is not necessary

q2: may be solved by satisfying dilution of 2nd; avoid to take the same species of lysate/homogenate and specificity of 2nd Ab

if there are still problems with the 2nd Ab, try to specify your Ab-antigen interaction by parallel testing with a block peptide (some companies provide the immunogenic peptide together with the primary Ab)

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What are the size of the contaminated bands? If they fall to either the size of light OR heavy chain, and that conincidently about the same size of your protein, choose 2nd Ab which only recognize the other chain (either Fc or Fab, depend on your need). Or another way, don't use the 2nd Ab such as goat-anti-rabbit-HRP, ... but use ProteinA-HRP, ProteinG-HRP or mix of the two.

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