Supernatant proceed to ammonium sulphate precipitation (standard method)
1) 0-20% (centrifuge 7000g x 20 min) -> 20%-50% (centrifuge 7000g x 20 min)
2) Procedure done in ice while stirring
3) Fine ammonium sulphate
4) supernatant was buffered with 50mM Tris-HCl pH7.5 prior experiment
5) (NH4)2SO4 was added slowly (17.55 g in 50ml ), fully added between 5-8hrs (white foam cannot be avoid)
6) Incubated at 4 ͦC chiller for 12hrs while stirring
Note: activity loss about 75%, fold less than 1
Q: do i need to add all the ammonium sulphate within certain range of time and how can i avoid formation of white foam (very little)?
Protocol seems OK but I miss some details. Where is your protein? in which supernatant or in which pellet. May be in both pellet and supernatant?
Also I miss removal of ammonium sulfate before the determination of your protein, unless you do a high dilution and then the amount of ammonium sulfate is low.
I understand that you are 5-8 hours for adding ammonium sulfate. Please note that ammonium fractionement is a "quick" procedure. You do not need to wait so long, and also it is not good to stir for so long.
the protein content of extracellular supernatant is 0.1mg/ml (on going experiment : increase the volume of culture to increase protein content)
Based on my reading its said that the protein content should be 1mg/ml before proceed to ammonium sulphate precipitation and time for adding (NH4)2SO4 is 2-10g/min (on going experiment). Is there anything that i should take into consideration?
If possible to be near 1 mg/ml better than 0.1 mg/ml.
How do you follow your protein? By ELISA?
Do you know already where your protein go agter adding a given amount of ammonium suldate (pellet or supernatant at a given % of saturation)?
i only did bradford and check activity with pnp assay...it go into pellet after 50%...
If I understand well, your protein is in the 50% pellet, nothing is in the 50% supernatant and then, in this 50% pellet, you have a reduction of 75% of original activity?
If so I would remove salt before doing the determination. Using dialysis or, better a pre-packed gel filtration column. For instance:
http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences/products/AlternativeProductStructure_17402/17085101
You might consider HIC chromatography in lieu of precipitation. But is the basic question concerning protein folding? If it's about folding: It may not matter how you purify the protein if you intend to refold as the next stage. Refolding usually begins with complete protein denaturation firstly followed by a partial factorial matrix to determine folding conditions. So if your purification results in denaturation, you would be denaturing the protein anyway to start refolds.
Run with standard proteins and determine the amount of proteins in your samples before electrophoresis
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