fluorescence in situ zymography


Hello All,

One other issue with this method to resolve, hopefully the last one!
I need some advice on the use of PMSF(phenylmethylsulphonylfluoride) in
solution. The solution contains:
50mmol Tris-HCl, pH 7.4,
10mmol CaCl2*2H2O,
0.05% Brij 35,
5mmol PMSF

Fluorescence-conjugated gelatin is then added to this solution and incubated
with tissue sections to detect gelatinase activity. As I understand it, PMSF
is added to inhibit any serine proteases which may be present in the tissue.
To get the PMSF to dissolve, the solution needs to be heated to almost
boiling. My first question is will the heating destroy the PMSF action? If
so how do I get it to dissolve another way, can it be dissolved in ethanol
for example? Will the addition of alcohol have any other effects on my

thanks in advance,

Abigail Mackey
Department Sport and Exercise Sciences
University of Limerick

tel +353 (0)61 20 28 84
fax +353 (0)61 33 04 31

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