I extracted total protein from agro-infiltered N. benthamiana leaves by using following extraction buffer :-
Protein Extraction Buffer- 50mM Tris/HCl pH 7.6, 100mM NaCl, 0.05% Tween20, 1mM EDTA, 1mM PMSF(phenylmethyl sulfonyl fluoride), 5ug/ml Leupeptin
as a result I got green colour supernatant, as expected. Now I want to know the best method to extract protein from that solution and in end i need a solution of proteins to check its concentration and work further.
Please help
Dear Partima
If you want to quantify proteins, you can use the Tris extract you have directly to do so. The green colour does not interefer with protein assay. If you are looking for something like enzyme assay, It would be better if you use HEPES (50 mM pH 7.4) containing EDTA and PMSF, because Tris inhibits some enzymes. You can use HEPES extract for anything including protein assay, enzyme assay, electrophoresis, etc.
The most important thing is to work in the cold and keep use samples frozen or if necessary on ice for very short time.
Good luck
try this (0.05 M Tris-HCl pH 8.0, 0.2% SDS, 5M Urea, 1% ß-mecaptoethanol), originaly meant for total seed proteins extraction however in our lab we have used it for rice and brassica leaves as well. take atleast 0.02g of leaves tissues extract. centrifuge @ 12000 rpm/10min. and the supernatent is all proteins.
Thanks Gaber Sir for your reply. I want to do western blot of that protein. if green color is not a big issue hopefully i get my protein. i will check and update.
Thanks Haris Khurshid .. if this time i will not get my protein. i will definitely try this buffer next time. my main concern is green color
Use 10 % TCA in equal proportion to your extract of green color, mix it, and allow to precipitate for 15 minutes, centrifuge it , then separate the supernatant, again treat the pellet with 5% TCA for 10 minutes again centrifuge the contents at 5000 rpm for 30 m inutes, now pool supernatant one and two and gow for Lowery test. You can go for further extraction after desaltation, dialysis and speed vac concentration. Now you can analyze your interested protein by SDS page, Southern blotting, ELISA and HPLC and and on affinity chromatography.
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