My protein is 43kDa. I want to use ammonium sulphate cut to precipate my protein. Can anyone suggest at what concentration saturation will I achieve precipitation of my protein? Further down the lane, I want to run my protein concentrate on gel filtration column to get a purified fraction of my protein. How should I minimize interference of other proteins in the total protein concentration? How do I get a clean fraction of my targeted protein?
The amount varies with the particular protein (note that the percentages usually given for this technique are % saturation, not wt/vol%, which is a much higher number). If you are using it as a purifying instead of using it as simply a concentrating step you can add it step wise (in 10% saturation increments) and remove the precipitated material after each addition.
http://web.mnstate.edu/provost/AmmoniumSulfateProtocol.pdf
http://blog.dilyx.com/2013/03/a-protein-purification-classic-ammonium.html
The amount varies with the particular protein (note that the percentages usually given for this technique are % saturation, not wt/vol%, which is a much higher number). If you are using it as a purifying instead of using it as simply a concentrating step you can add it step wise (in 10% saturation increments) and remove the precipitated material after each addition.
http://web.mnstate.edu/provost/AmmoniumSulfateProtocol.pdf
http://blog.dilyx.com/2013/03/a-protein-purification-classic-ammonium.html
Keep in mind that you will lose some of the protein in the fractionation process and that traces of the ammonium sulfate can be difficult to get rid of. An ion exchange-gel filtration column combination may be more efficient if your protein is relatively clean.
You can sequantial fractionation by size exclusion chromatograghy, detrmine the fraction that includes your protein of interest, and then using cation or anion chromatography to get your protein purified.
This is the simple repeatable technique I love, thus it makes me happy you want to try it! Dr. Jeffrey Brender already told you everything I agree, please follow his advices. The precipitate you can resuspent in any native buffer you want, the rest over of ammonium sulfate you dialyze. I have never seen such an easy protein pellets to rehydrate like in this technique. Keep fingers cross:)
Follow the method described by Mohammad...
Hi Amruta,
Ammonium sulphate method is a reasonablle method for concentration and or enrichment of proteins.
The % used for protein saturation varies from protein to protein and has little to do with size rather than protein amino acid composition and its structure.
I suggest as Jeffrey already suggested doing a small scale pre- trial with 10% increments to determine the right % A.S to use for your protein.
After you determine the best % A.S for use you can do a single concentration precipitation with a large batch of protein.
As for the second part of the size exclusion, depending on how well purified you would need your protein:
If protein purification can stand a few impurities then it should suffice
If you need a highly purified protein, the two steps you mentioned will (in most cases) not be enough, so I would suggest adding another enrichment step such as ion exchange (of other ).
Hi Amrutha,
Ammonium sulfate precipitation is a simple and efficient technique to remove unwanted protein and concentrate our protein of interest. Please consider the important things as said by Dr. Jeffery. Its not the % of the solution its the saturation level and kindly use the precipiation table available elsewhere. Do not discard the precipitate or supernatant at any stage and try a SDS PAGE of all the protein at every stage of precipitation. U dont want to miss any of ur protein. Of course you will lose a certain quantity of your protein, having a high initial protein concentration is advised.
Another issue is the salt removal, do dialyse the sample well after precipitating to get rid of the ammonium sulfate.
Gud luck.
Thank you everyone for the helpful replies. I will update you soon with the experiment. Thanks!
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