Does anybody familiarize with the freeze thawing method to isolate proteins? what is the normal yield of protein we can get from it?
Suspend your cells in buffer with protease inhibitors and place the tube on ice until the dry ice bath is ready. Freeze-thaw your cells using the bath of dry ice and ethanol. Hold your tube of cells down in the bath until completely frozen, then thaw them. Repeat the freeze-thaw 3 times. Pellet the large cell debris and unbroken cells at 500xg for 5 min and the supernatant should contain all your total protein non-denatured. The amount of protein will vary per cell type and how many cells you use. The more the better!
We extract crude Leishmania proteins using freeze-thawing in liquid nitrogen. Wash the cells in PBS and centrifuge at 4oC. Then, resuspend the cells (pellet) with PBS in the presence of cocktails of protease inhibitors and snap freeze in liquid nitrogen and thaw in ice. Repeat the freeze thawing three to five times. You can also sonicate it after the freeze thawing. Good luck!
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