Dear all,
I want to precipitate protein from supernatant of bacteria in culture and my question is whenever i do ammonium sulfate fraction i get floating pellet but, not getting settled pellet. what wrong in my protocol, I reach 80 percent amonium sulfate saturation witch normally any protein precipitate at this point.
Does anyone have an idea about this?
Is your bacterial growth medium unusually dense? For example, does it contain a lot of sugar or glycerol?
80% saturation ammonium sulfate shos a density about 1.2 g/ml. With this density and salt concentration, a lot of proteins are insoluble, but they floculate since the density is lower than 1.2.
On the other hand, a few proteins are still soluble at this high salt concentration, even 100%. I remember the S-100 neuronal protein (its name means soluble at 100% ammonium sulfate)..
Dear ALL,
Yes Professor Addam THE GROWTH medium of my bacteria contain 10 gr glycerol and 10 gr Glucose . Where is the problem ?
Dear Pr solano ,
thank u very much , AS i understand from your reponse , I have to reduce the quantity of amonium sulfate to get more precipitate proteins (60 percent for exmple).
you don t think that the room temperture may affect protein preciptaion ?
Best regards
as indicated some proteins will remain in solution at 80% AmSO4 - but that is not a problem - just remove the settled and floating pellets (the later by filtration maybe) and dialyse the liquid fraction and you will have remove all proteins not soluble at 80% - that should give you a very good fractionation.
My thought was that if the growth medium was supplemented with glycerol and/or sugar, the density might be high enough that the ammonium sulfate-precipitated protein would float instead of pellet. Since you didn't specify the volume of medium in which 10 g of glycerol and 10 g of glucose were added, I can't tell how much the density was increased by them. In any case, Francisco Solano pointed out that 80% ammonium sulfate itself may have a high enough density to keep the precipitate from pelleting.
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