cleavable acrylamide cross-linkers?

Klaus.Matthaei at anu.edu.au Klaus.Matthaei at anu.edu.au
Fri Oct 8 18:06:43 EST 1993


>Hi Bionetters:
>
>        I need to purify a 210 kDa protein.  It need not be 100% pure, but
>as pure as I can get it.  I have been immunoprecipitating and then running
>the precipitate on a 7.5% acrylamide gel.  This works fine except that my
>recovery by electroelution from the gel nets me only 20% of the band of
>interest.  If I load two lanes with identical ammounts, cut out the band of
>interest in each lane, electroelute the protein from one and save the
>other; I find that I only retreive 20% of the CPM in the eluate, the other
>80% remains in the gel.
>
>        I know that there are some other cross-linkers for acrylamide. 
>Are any of them cleavable?  I'd think it would be handy to "digest" the
>gel and thus find it easier to get the protein out of it.  Alternatively,
>perhaps I could use agarose instead of acrylamide, but I have no
>experience with agarose and proteins.
>
>        Any help is greatly appreciated.
>
>--
>********************************************************************
>*  Brian Foley               *     If we knew what we were doing   *
>*  Molecular Genetics Dept.  *     it wouldn't be called research  *
>*  University of Vermont     *                                     

Dear Brian

FROM ANOTHER TIME WHEN I WAS MORE A BIOCHEMIST>
If your protein has no disulphide bonds you can use BAC as the crosslinker
instead of BIS in the acrylamide.  The gel then dissolves in
mercaptoethanol to recover your protein.  See p297 of your 1993 Biorad
cattle-dog.

Cheers, Klaus
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Klaus Matthaei
Gene Targeting
The John Curtin School of Medical Research
The Australian National University
E-mail: Klaus.Matthaei at anu.edu.au

"Think twice - Do once"
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