Help!!! need a protein isolation protocol from leaves

J. Norrie 4104jnor at vm1.ulaval.ca
Thu Oct 12 10:17:50 EST 1995


Dear Friends,  
 I am interested in isolating my protein of interest from tomato leaves by
using an affinity column.
My extraction buffer includes beta-mercaptoethanol, DTT, ethylene glycol,
Magnesium chloride, EDTA, 2% PVPP, 1% Dowex, citrate-phosphate pH6, and
PMSF. The soluble proteins are ppted with 80% amm.sulfate, proteins
dissolved in cit-phos buffer, the trace amm.sulphate is removed by passing
thro' sephadex G-25. 
The protein aliquoted into 1ml and stored at -20deg. The problem starts
here after. After I thaw the protein samples, invariably I get the ppt.
The supernatent, when checked for the protein concentration shows nearly
50% loss (as compared to the protein samples befor freezing i.e when the
samples are clear), when analysed by Bradford reagent. Could someone
suggest me the solution and also give me a reason why proteins ppt. after
freezing? The pptation of proteins  is also seen when samples are kept on
ice for a long time.
Are the phenols responsible for this, or the lipids(?), well somebody gave
me lipids as the reason..am not quite sure about it. I would be grateful
to receive all the comments and suggestions. Thanking in advance,
Savita
email: 4104vsav at vm1.ulaval.ca



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