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protein purification - (Sep/15/2005 )


I have a problem with isolation of a ser/thr phosphatase from a plant Brassica. The same protein in animal system has been well chracterized and its antibody is also available. But the same antibody (polyclonal) does not work in my system. How should I go for detection of the protein in my samples. I can estimate enzyme activity using an assay but cannot purify the protein to homogeneity since I do not know its exact size (no signal with western blotting). How should I isolate this protein??
Somebody plz help with this



I had the same problem. Antibody directed against protein produced in baculovirus, did not want to react with the same protein produced in plants.
I was told that it is a common problem, as often antibodies are directed against the sugars on protein, and ways of glycosilation is quite different in plants and baculovirus.

Also, plant protein extract - it is an another problem. There are probably something in it which prevent good interaction with antibodies. I quitted that lab and the problem was still not resolved.



is it possible to clone it?


Others things:
Plant cells are highly compartemented; in most cases the bulk of the volume is vacuolar space, which can be filled with quite acid solution, proteases and other detrimental compounds. There is also a large amount of cell wall (cellulose), and the chloroplasts, starch granules and other organelles occupy much of the cytoplasmic space. Indeed the cytoplasm may often be no more than 1-2% of total cell volume in plants. Consequently plant extract may be very low in protein even if very little fluid was added when making the extract