I tried extracting my cartilaginous tissue sample with the help of RIPA buffer, precipitated it with the help of acetone: methanol (8:1) volume to volume of sample. Tried dissolving it with CTAB buffer (1M tris, 4M Nacl, 2%CTAB with inhibitors) to remove residual polysaccharides.
But my pellet is not dissolving, what can be done to dissolve it?
After this i would need to re-precipitate it with acetone: methanol (8:1) prior to 2DE.
Can anybody suggest the best way??
Hi Sharon Miracle Nayagam . When you precipitate RIPA buffer with acetone/methanol you are also precipitating the detergents in the RIPA buffer, including SDS. At high concentrations CTAB and SDS form insoluble precipitates.
Chapter Interaction of Cetyltrimethylammonium Bromide with Sodium Do...
I would recommend using the RIPA extract straight or dissolving the acetone methanol RIPA pellet in diH2O for the 2D.
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