Problem extracting protein from plant leaf - (May/27/2006 )

Hello,

I have prepared crude extract from dried leaves with 0.1 M phosphate buffer containing 0.1% B-mercaptoethanol and 2mM PMSF. The cell free extract is red brown in color. When I dilute the crude extract with the same phosphate buffer, I get turbidity and am not sure whether it's protein precipitating or something else. What is causing this? Somewhere I read that dilution of buffer causes aggregation of proteins. Is this it? As a matter of fact, it is not happening with buffer of other pH. When I add TCA to it, the turbiditiy disappears.

Best Regards,
Rigzin

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I have sorted out the above problem. But the problem I am facing right now is the low amount of proteins being extracted from the leaves. From Bradford assay I found out that my protein content is just 8 ug per 100 ul. The extraction buffer is the same as above with the addition of PVPP and the ratio of weight of the leaves to the volume of the buffer is 1:4. But due to the absorption of the liquid by the leaves, I have to add more buffer about 1 gm of leaves in 10 ml. So my question is should I use lesser volume of buffer to concentrate the protein or should I use harsher extraction buffer? Or, can it be possible that the proteases are degrading the proteins out there. Ironically I am trying to characterize proteases in this leaves of this plant I m working on.

Best Regards,
Rigzin

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