protein precipitation - (Aug/18/2008 )
Hello, I am trying to do an ammonium sulfate precip of a solubilized protein solution. I have made a 100% saturated ammoinium sulfate (AS) solution in my desired buffer (HBSS + 20mg/ml chaps). I then incubated overnight my sample with 20, 40, 60 or 80 % AS. I see precipitate but am unable to pellett this. I have tried spinning at 1500 xg for 15 min, 30 000 x g for 15 min and even 50 000 x g for 1 hour. My precipitate all reamians on the top of the liquid in the 80% and only ends up in a very unstable pellet about 1/2 way down the tube in the 20% sat solution. Any ideas of where i am going wrong here? thank you.
Do you have enough protein in the solution? Use the highest conc. you have available and add the ammon sul dropwise in the cold. ratio of protein sol to amm sul is 1:1
my protocol was as follows
10 ml sample + 40 ml Ammonium sulfate (AS) = 80%
10 ml sample + 20 ml AS = 60 %
10 ml sample + 7 ml AS = 40 %
10 ml sample + 2.5 ml AS = 20%
I was not using a concentrated sample, but i could definatly see protein precipitating. Are these volumes too large? I dont see how i can keep a 1:1 ratio when i want different final concentrations of AS, the way i see it, i using a 1:1 will only allow me to go 50% sat at the highest.