Hi Tamsin,

> (2a) Grind the the frozen material using a grinding buffer; something
> like ice-cold TRIS-HCl (0.2 M, pH 8.5; 0.25 M sucrose; 1.0 mM
> EDTA; 0.25 mM phenylmethylsulfonyl fluoride; 0.1 mM MgCl2).
> Rule of thumb: for 500 mg of plant material use 2 x 0.75 mL of
> grinding buffer (i.e. in two steps). By way of explanation, the EDTA
> and PMSF are used to switch off protease activity.

You probably know that these protease inhibitors have the half time
of a gnat when in water so it is best to make them up in alcohol and
add them just after the extraction buffer hits the plant material.

They are meant to be irreversible..

Yours


Andrew




Dr A.M.Johnston
SPD
Scottish Crop Research Institute
Invergowrie
Dundee
DD2 5DA
Scotland

Tel No: (44) 1382 562731 ext 3732
Fax No: (44) 1382 562426