beta galactosidase purification

Bipin K Dalmia bipin at iastate.edu
Thu May 12 17:21:15 EST 1994


In article <1994May9.102353.1 at pearl.tufts.edu>,
 <rfeldber at pearl.tufts.edu> wrote:
>Netters;
>	I have been asked to put together a section of a teaching lab which
>will demonstrate enzyme purification/characterization using the beta
>galactosidase of E. coli. Does anyone already have a simple, fool-proof
>protocol for partial purification of this enzyme? I was thinking of asking
>different groups of students to use different protocols and then compare
>specific activities and gel patterns. Any suggestions would be greatfully
>appreciated.

the method of steers (i can dig up the reference if you don't have it)
has always worked for me. just buy beta-d-thiogalactopyranoside agarose
from sigma, pack it in a column, run the mixture of proteins with b-gal
at around pH 7. b-gal will bind very specifically. wash off everything
else and elute with 100 mM sodium borate pH 10.00. minimize the exposure
to pH 10 by immediately dialyzing or do what i used to do, elute in a
equal volume of 1 M tris-hcl pH 7.0. this would dilute your enzyme but
would recover almost 100 % of the activity. you may already know this,
but b-gal needs 10 mM MgCl2 and 10 mM 2-mercaptoethanol in all buffers
to be stable.

bip

-- 
bipin k. dalmia               the other night i was lying on my bed, looking
bipin at iastate.edu             up at the beautiful stars, and i said to myself, 
n2.bkd at isumvs.iastate.edu     'where the F*CK is my ROOF !!'
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