I want to use my enzyme in detergent so i am going for partial purification of enzyme, i am confuse that which method is appropriate for me ammonium sulphate precipitation or acetone precipitation? What is difference in both method related to its efficiency?
Before any comment or suggestion I want to ask some question. Which substrate are you using for your partially purified enzyme? natural or synthetic? Why you thinking precipitation method to purify?
I think you can use size exclusion chromatography first if you know about the size enzyme. Then you can use 2nd step purification. The precipitated enzyme might not be active. Good luck..
I am using natural substrate(chicken feathers) for my enzyme(keratinase). and I have to do optimization of enzyme after precipitation, Its commonly used method so i wish to do enzyme purification by precipitation. Thanks for your reply M G Sharoar sir.
Do not expect high purification fold after salt/acetone precipitation. I would rather consider it as concentration rather than purification step.
I'm not sure about difference in efficiency, but acetone precipitation may result in inactivation, while ammonium sulphate should have rather protective effect.
I am also agree with Tomas. Use ammonium sulfate precipitation for it, acetone is more harsh, yr protein may lose activity. But it depends, if you have access to protein purification system (FPLC), use ion exchange for first step purification (anion-exchanger), if PI of yr protein is acidic it will bind to resin, if basic it will not bind and then other contaminants will bind. if later is the case concentrate flow through, and if before elute column with NaCl gradient. after that pool your activity fraction and inject in gel filtration. this could be the best way to purify the protein for you.
ammonium sulphate pp is usually used as one of the first steps in a purification (pre-chromatographic steps), you can try 20-30-40% SA and analyse in a SDS-PAGE where is your protein, but if you feed is natural, maybe you will need to follow the purification by meassuring activity or by western blot. Afterwards, if your enzyme is in the pellet maybe you can use as a capture step a IEX ( anion or cation exchange chromatography) but if your protein is in the sob, a HIC (ej: phenil sepharose) is very useful. I would use gel filtration as polishing step (final step) because you have to use very small volumes.
Every protein is different and particular and the purification procedure is more difficult if your feed is natural
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