Protein Binding Studies on Cells

S. McKay S.Mckay at sct.gu.edu.au
Thu Dec 10 00:06:34 EST 1998


Hi,

        I'm about to embark on some binding studies with a 12kDa
S35-labelled protein on several monolayer and suspension cell cultures.  I
am adapting a previously published protocol for this protein but labelled
with I125.  The protocol indicates that cells were washed with PBS
containing 1mM MgCl2 and 1mM CaCl2.  The I125-protein was incubated in DMEM
containing 0.5% BSA and 25mM HEPES (cells were normally grown in DMEM +
0.5% FBS for experiments and 10% FBS for culturing).

        I was wondering if the wash with MgCl2 and CaCl2 was some type of
way to enhance membrane binding and what the purpose of the BSA and
aditional HEPES was in the experimental media.  It seems odd to add extra
buffering capacity for experiments which only run for 2-20hrs.

Any enlightenment would be much appreciated.

S.McKay





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