Basic Question: How does Western blot work?

Carl Simard c.simard at sympatico.ca
Wed Oct 21 19:51:12 EST 1998


You're right on many point. However, with a western, you usually have a
dnatured protein on your membrane. If I understand your question well, you ask
why a polyclonal serum made against a denatured protein will recognise a
native protein?

    When you inject your denatured protein in the rabbit, the rabbit raise an
immune response against many epitopes (or many regions) of the protein. A
antibody in your serum, for example, may bind to a specific sequence of five
amino acids while another will bind another region. So, in your serum, you
will have a mix of antibodies to recognise all of the diverse epitope of the
denatured proteins. Many of these epitopes will also be present in the native
protein ans your polyclonal serum will bind to them. Evidently, all of the
antibodies in your polyclonal serum will not bind the native protein, but the
amount will be largely enough to give a signal.

    If you used monoclonal antibody instead of polyclonal, you will face a
problem. If you direct your monoclonal antibody against an epitope of the
denatured protein that is not presented by the native protein (for example, a
domain inside a globular protein), your antibody will just work with the
denatured protein.

Hope these clarifications help you!
Carl Simard
c.simard at sympatico.ca

Tristan wrote:

> Hello everyone:
>
>    I have some questions and about how Ab recognizes Ag during a
> PAGE-Western blot process.
>
>    My polyclonal Ab was produced by injecting the protein PAGE gel slice
> into a rabbit (I guess, because we sent the slice to a company to make the
> Ab).  I always assume that after PAGE, the protein is denatured and lose
> its 2nd, 3rd structure, especially after boiling.  So the Ab is to
> recognized a denatured protein (or should I say a long polypeptide?)  But
> why can the same Ab sometimes also be used to detect native protein such
> as in immunohisochemistry?  Does the Ab recognize a specific short length
> of peptide, or does it recognize the specific structure of a native
> protein?
>
>    One professor here told me that the washing process after PAGE and
> during the blocking step is to renature the protein so that Ab can
> recognize it.  Does it means that most proteins can refold to their native
> shape even after boiling and SDS saturation, and Ab is actually recognize
> native protein?
>
>    Thank you very much for your help.
>
> Tristan
> AECOM




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