I am planning to isolate total proteins of gram positive bacteria. At final stage, I will resuspend the pellet in protein sample buffer that includes 40 mM Tris/HCl (pH 8) , 4 mM EDTA, 8% SDS, 40% Glycerol and sonicate it. Is it convenient for total protein isolation or should I change the buffer for better efficiency?
I would include NaCl (150mM or more) to maintain the ionic strength. and glycerol seems a bit higher to me, normally we use 10% at max. In addition you can consider adding PMSF/protease inhibitor cocktail to kill the proteases. edta and sds are kept to be stringent since it may disturb the protein you are interested in!
I was using similiar protocol and generaly it works.
Sonication is rather crucial step contributing to extraction efficiency.
You are going to run a SDS-PAGE electrphoresis after extraction, right?
I am also planning to extract total proteins from Ralstonia solanacearum for 2D analysis. What should be sonication conditions for total proteins? I mean not only for soluble proteins but also for membrane or nuclei proteins.
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