Problem in acetone precipitation of plant proteins. - (May/28/2013 )
I extracted proteins from etiolated seedlings. I do a 70% ammonium sulphate precipitation. Redissolve my sample and then do a 60% acetone precipitation at -20 for 1 hour and then centrifuge. I start of with 3 g of tissues. After acetone precipitation I can see white pellet at the bottom which is the protein and it dissolves easily too. The problem is along with the white pellet I get a block of another pellet too at the bottom which is pretty big and compact and looks different from the white pellet, I mentioned earlier and it does not dissolve.I vortex it and once I keep my tubes for sometime it forms pellet at the bottom again. I have cytoplasmic, cell wall and membrane proteins. In all three fractions this happens. After dissolving my proteins I centrifuge and with a lot of effort get clear solution. The cell-wall one is the hardest to remove this pellet. I am losing a lot of proteins in this way. I still dissolve that pellet as I think it contains some proteins too.
Any suggestions as why this is happening and how to improve is much appreciated. I don't dry my pellet a long time. I keep smelling the tubes and once I cannot smell the acetone I add my resuspending buffer.
after you resuspend the ammonium sulfate pellet, do you dialyze away residual salt? you may be seeing a salt pellet.
check the pellet on a gel to see if it is carrying your proteins with it.