Counting radioactive proteins in yeast?

Fergus Doherty mbxfd at unix.ccc.nottingham.ac.uk
Thu Dec 12 04:52:27 EST 1996


I am attempting pulse-chase experiments with yeast labelled with
35S-methionine.  I need to determine the total amount of cell associated
radioactivity in protein at zero time of the chase.  I precipitate proteins
with TCA (final conc 10%), by sonicating the cells in TCA, recover the
precipitated protein and attempt to dissolve it in either NaOH (5M) or neat
formic acid by sonication. However the results are highly variable and 	 am
not convinced all the precipitated protein has dissolved. Certainly there
is a lot of insoluble material left, which I assume is the cell wall.  Has
anyone tried this and can suggest a reliable way to recover all the TCA
precipitated radiolabelled protein from the yeast in soluble form for
scintillation counting?

Replies direct to me please.


Fergus Doherty,
dept. Biochemistry,
University Medical School,
Queen's Medical Centre,
Nottingham NG7 2UH
Tel: (0)115 970 9366  FAX (0)115 942 2225 Internet:
Fergus.Doherty at nottingham.ac.uk



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