Recovery of protein from Polyacrilamide Gels

engelbert_buxbaum4 at my-deja.com engelbert_buxbaum4 at my-deja.com
Tue Dec 12 01:39:01 EST 2000


In article <004a01c063d6$4d63a120$55cab0c8 at computer>,
  nathan. at zaz.com.br ("Nathanael Pinheiro") wrote:
> Does anyone have any experience or information about how may I extract
> a 21KDa protein from an SDS-PAGE without any "electroelution
> apparatus"?

That would depend on what you want to do with the protein. One of the
possibilities is to digest the gel. If bisacrylamide is used as
crosslinker, that is likely to do some damage to the protein, but there
are crosslinkers available with an S-S bridge that can be digested with
mercaptoethanol (Pierce is one of the suppliers).

With a small protein it might be possible to vacuum-elute, but you'd
have to build suitable equipment. Some of the protein may also be
extractable simply by mixing the finely mushed gel with buffer in an
end-over-end mixer over night, however, do not expect complete recovery.

If you want the protein for antibody production, no elution is
necessary. Stain and destain the gel with CBB as usual, this will at the
same time remove any traces of unpolymerised acrylamide (which would be
toxic to the animal). Cut out your band, swell in distilled water and
mush the gel by passing through a 27G needle, then mix with Freund's
adjuvant and inject. The gel slows the release of the protein and acts
as a mild adjuvant, improving antigenicity.


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