My media contains starch (1% w/v) in LB. But the problem is that the starch is also getting precipitated by during ammonium sulphate precipitation (60% w/v). Can anyone suggest how to prevent the precipitation of starch or suggest any other methods of precipitation that does not precipitate starch?
Starch granules should sediment without ammonium sulfate, so I suggest before you add the ammonium sulfate (to precipitate a certain protein I assume), spin the medium at 5000g for 5 minutes, transfer the supernatant, and then add the ammonium sulfate, this should reduce the amount of starch in your pellets. Whenever you do ammonium sulfate precipitations of proteins, it helps to establish foirst at what concentration the protein of interest precipitates. Try small scale samples in Eppendorf tubes (20%, 25%, 30% etc.. up to 60% ammonium sulfate) and incubate 2 hours on ice and then spin at 4 degrees. Check at what concentration the protein of interest is precipitated. May be the starch precipitates at a different concentration and then you can fractionate differentially.
Sir, i grow the microbe producing cutinase in the LB media. I centrifuge at 10000 rpm for 15 min to seperate the cells. Later, when ammonium sulphate is added the starch still gets precipitated...
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