Basic Question: How does Western blot work?
svetlov at oncology.wisc.edu
Fri Oct 23 00:40:08 EST 1998
In article <fengli-2110980830450001 at skylark.aecom.yu.edu>,
fengli at aecom.yu.edu (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
Most of the protein epitopes are relatively short ca. 10 a. a. and the
determinants that responsible for most of the affinity are usually 3-4 a.
a. out of these 10. If this epitope is on the surface of the native protein
the Ab would recognize it and likely to supershift or precipitate the
native protein. In many cases so called conformation-sensitive antibodies
actually recognize not the conformation per se but the surface epitopes
whose presentation is affected by conformational changes.
> 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?
Yes, indeed. This is the basis of such techniques as South-Western and
Far-Western where the protein after transfer to membrane is washed to
remove the denaturant and incubated in smth. as simple as blotto (or in
more sophisticated "refolding" solution featuring TFE, Arg etc.) to allow
for refolding. Usually one can achieve enough refolding to allow specific
recognition by a DNA (S-W) or protein (F-W) probe. The problem is that
immobilization on membrane interferes with refolding: in several cases
incl. recent report from our lab the minimal size of a polypeptide
effectively refolding a protein-binding site is significantly larger when
the polypeptide is refolded on the nitrocellulose membrane than when it is
refolded in solution or immobilized on Ni-NTA column.
Hope this helps.
Vladimir Svetlov, PhD
McArdle Lab for Cancer Res.
1400 University Ave.
Madison, WI 53706
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