V8 protease/domain mapping

marder at agri.huji.ac.il marder at agri.huji.ac.il
Fri Feb 19 06:52:58 EST 1993


In article <1993Feb16.094451.1 at molbiol.ox.ac.uk>, rpgrant at molbiol.ox.ac.uk writes:
> Hi all!
>
> I'm thinking of using V8 protease to investigate the domain structure
> of a large-ish protein (280 kDa).  Does anyone know of any good
> protocols/sources?
>
> Thanks.
> --
> Richard P. Grant                        rpgrant at molbiol.ox.ac.uk
> Chemical Pathology Department           Tel. +44 865 275565
> Sir William Dunn School of Pathology    Fax. +44 865 275556
> OXFORD, UK.				<><
A convenient method is by "Cleveland et al. (1977) J. Biol. Chem. 252,
1102-1108."  The protein is extracted as a band from a gel, re-equilibrated
with buffer, placed directly on a second gel and overlayed with protease.
Digestion occurs in the stack of the second gel.

I have used this method extensively with V8 protease - it works fine in
0.1% SDS and normal gel conditions.  V8 is available from many biochemical
companies (sometime called endoproteinase Glu-C).

--
                                 '      Jonathan B. Marder
Internet: MARDER at AGRI.HUJI.AC.IL |      Department of Agricultural Botany
Bitnet:   MARDER at HUJIAGRI        | /\/  The Hebrew University of Jerusalem
Phone:    (08 or +9728) 481918   |/  \  Faculty of Agriculture
Fax:      (08 or +9728) 467763   /      P.O.Box 12, Rehovot 76100, ISRAEL



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