I wanna extract my expected protein which is targeted to nuclear in plant system. May I really need to add any detergent such as SDS or triton X100 in extraction buffer? Does PBS buffer is good to use for protein extraction? And how you suggest to use PBS buffer for better extract? Could you Please suggest any good protocol to best extraction? I need to extract this protein to purify by nickel column.
Dear Maria
You may find inspiration in this previous RG post from Kyle W. Bender, University of Illinois. Kyle posted
Depends on your needs, but the extraction buffer that I routinely use for protein purifications from plants (i.e. immunopurifications) is as follows:
50 mM Tris-HCl, pH 7.5
100 mM NaCl (can swap with KCl)
250 mM Mannitol
5 mM EDTA
1 mM DTT – add fresh just before you start
10 % glycerol
Plant protease inhibitor cocktail (from Sigma Aldrich)
Glycerol and mannitol in this recipe are optional as well (they aid in stabilization of proteins).
Which lysis buffer/ method do you suggest for total protein extraction from plant cell suspension culture? - ResearchGate. Available from: https://www.researchgate.net/post/Which_lysis_buffer_method_do_you_suggest_for_total_protein_extraction_from_plant_cell_suspension_culture [accessed Jan 30, 2017].
BR Birger
Dear Briger Brodin
thanks for your attention
But I wan to use nickle column so I should dialysis EDTA and DTT in buffer extraction protein.
Ahhh, yeah thats off course tricky..another protocol is mentioned in the thread above (by Boon), copyed below (no EDTA but still DTT);
"There are many extraction protocols. If for 2-D analysis, we used lysis buffer (7 M Urea, 2 M thiourea, 4% CHAPS, 100 mM DTT, 40 mM Tris-HCl, pH8.5, protease inhibitor cocktail). Then, centrifuge at 14000 xg for 20 min at 4C.
You might need to increase the amount of starting material (cells) in order to get higher yield."
You may play around with these as starting points, but replace DTT with the weaker beta-mercaptoethanol or TCEP (expensive , though)
If you work with cell cultures that buffuer is OK. If you are working with plant tissues, you may need some detergent ranging from 0.1% to 1% of Triton X-100.
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