Hi,
I want to extract total protein from tobacco suspension culture and used TCA- acetone method which I think was not well for my analysis as the proteins are not solidified well in the sample buffer. I would like to use a lysis buffer to do the extraction. if you have any idea/ protoco , etc, I appreciate that.
best,
Hi Mahbobeh
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).
Hope that helps!
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.
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