Extracting 35S-labelled Protein from E.coli

S. McKay S.Mckay at sct.gu.edu.au
Fri Nov 27 19:06:35 EST 1998


Hi,

        I have a 12kDa protein plasmid which I have been expressing in
E.coli BL21.  This is a rountine procedure in our laboratory.  Extraction
of the protein is via sonification, 3 x 20" pulses at 20 microns. This is
ordinarily sufficient to lyse 2g of cells.  However, I have now been
attempting to label this protein with 35S-Met/Cys. The E.coli are grown in
a minimal media, induced and labelled before harvesting.  I have a total
wet cell weight of 0.14g from 60mls of culture.  This was resuspended in
2.8mls of lysis buffer (0.05M NH4 Acetate, 1mM EDTA, 0.5mM DTT and 5mM
Benzamidine) prior to sonification.

        Fluorography on test samples and beta-counting shows that even
after 11 pulses and several freeze thaw steps the majority of my protein
remains within the bacterial pellet and not in the supernatant.  Under
ordinary expression conditions (L-broth not minimal media) this protein
does not form inclusion bodies within the bacteria.

        I am becoming frustrated since this is usually a straight forward
procedure for the unlabelled protein and meanwhile my specific activity
will decrease daily.  Any suggests you may have would be greatfully
accepted.

S.McKay





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