I am using the extraction buffer for plant total protein isolation for MALDI. the composition of te buffer is Na- Phosphate buffer (PH 6), 1mM EDTA, glcerol, tritron x, PVPP, 2-Mercaptoethanol, PMSF and protease inhibitor cocktail. is it necessary to add sucrose in it? or is their any suggestion for ideal composition? can I use anything instead of PVPP (Polyvinyl poly pyrrolidone)?
PVPP and sucrose are necessary. This is a great commercial product you can use. I did get great answers
https://www.thermofisher.com/nl/en/home/life-science/protein-biology/protein-purification-isolation/cell-lysis-fractionation/cell-lysis-total-protein-extraction.html
PVPP is used to protect the proteins from polyphenols. You could use other reagents that bind polyphenols, e.g. polyethylene glycol.
Thank u all. I have RIPA buffer from Thermofisher.. is it appropriate for plant total protein extraction?
RIPA from Thermofisher contains different strength of detergent (NP40, SDS..). Strong RIPA is proper for total plant isolation if the detergent has no deleterious influence in your following experiments. You can find the product instruction of RIPA from the Thermofisher. I my experiments, I think the buffer (50 mM Tris–HCl pH 7.5, 100–150 mM NaCl, 10 % glycerol, 5 mM DTT, 1mM EDTA, Protease inhibitor cocktail (P9599, Sigma), 0.1%-1.5% tritonX-100(or NP40) is sufficient for total protein isolation. DTT and cocktail are properly added before use. The concentration of NaCl and the detergent are added depending on your purpose. If you want use RIPA buffer, additional protease inhibitor is required to added. Hope this can help you.
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