I'm doing precipitation with concentration of ammonium sulfate from 0-30%, 0-40% and so on until 0-90%. But, when I'm trying to centrifuge the precipitation result it always producing zero pellet of enzyme. I'm doing it with 8000 rpm for 20 min of centrifugation. Anyone know why does it happened?
Thank you
Dear Septian Yoesafat,
For how long do you leave your samples for precipitation? Maybe you should increase the time?
@Gabriele I leave it for a day (24 hours exactly) until the ammonium sulfate completely dissolved
Have you measured your protein concentration before doing ammonium sulfate precipitation? Measure also the concentration of your supernatant after adding ammonium sulfate to see whether the protein is still in solution or not.
Hi,
One of the critical points to cosider when doing salting-out is the concentration per mL of your protein. Diluted protein usually precipitates very little. So if your protein is diluted you may need to concentrate it by lyophilization or concentration device depending on your protein volume. Secondly run the precipitation when your protein sample is very cold and do the addition of ammonium sulphate pinch-wise over at least 30min, leave your sample to equilibrate for another 30 min under stirring. Then later allow your sample at 4C for at least 2hours. Centrifuge at high speed for 30min.
Hope this will help!
Hi Septian
You should have an idea about the [C] of your starting solution. Try to quantify by colorimetric methods and if you have low concentration try to concentrate by ultrafiltration considering the cutoff of the membrane according to the MW of your proteins of interest.
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