asking about the ammonium sulphate precipitation - (Sep/13/2006 )

Dear all,

I need to produce amylase immobilized from bacillus subtilis. Before that, i have to do ammonium sulphate precipitation. I add the ammonium sulphate 4.5g (30 saturated) into 20ml of amylase produced at 4 degree celcius and centrifuge it. But, there is no pellet found and I can't proceed to immobilization. I am very worry as i have to finish my project in one month. I need some advice. Thanks


Regards,
Elisa

try to increase centrifugation rate or
can't u change ammonium sulphate amount?
how many times u did precipitation? may be u don't need to do it anymore if all contaminant proteins were already removed..

how long do you precipitate for ?? might be worth extending the precipitation time as well as increasing the centrifugation speed and time (13,000 rpm for 30 min at 4degrees) as mentioned by a_sar

enjoy

Unless the protocol is written in stone, you might need to play around with it a bit. Clearly you have not reached the correct saturation level to precipitate your protein. Increase in small steps, while noting what percentage you are going to, and see what percent NH4SO4 does bring down the protein.

Sometimes you've just gotta do the expt again, that's why they call it re-search...

Unless the protocol is written in stone, you might need to play around with it a bit. Clearly you have not reached the correct saturation level to precipitate your protein. Increase in small steps, while noting what percentage you are going to, and see what percent NH4SO4 does bring down the protein.

Sometimes you've just gotta do the expt again, that's why they call it re-search...