Dear all,
I am using ammonium sulfate precipitation for my assay and my protein started to precipitate at 50%.
Therefore, is 50% ammonium sulfate considered concentrated?
Thank you very much
Hi,
How do you know the precipitates contained your protein of interest? Western blot?
As the earlier post indicated-did you detect your protein via Western blot or some activity assay method?
When using ammonium sulfate to precipitate proteins as a step in protein purification or for storage of the protein, the concentration of ammonium sulfate is often expressed as % of saturation. If that is what you mean by 50%, then you added 29.5 grams of ammonium sulfate per 100 ml of solution*. This is about 2 molar. This is enough to precipitate many, but not all, proteins. If you purchase proteins supplied as ammonium sulfate precipitates, they are often in 3 to 3.2 M ammonium sulfate.
*Equivalently you may have combined equal volumes of protein solution and saturated ammonium sulfate.
Thank you for all the answers, and yes, I detected my protein via western blot.
Adam's answer was very clear
Thank you!
the concentration at which the protein starts to precipitate, depends on the nature of the intended protein. If your sample starts precipitation at this point and several repetitions show the same result, so it may be the correct point.
We do ammonium sulfate fraction of protein by adding ammonium sulphate in %like 20_40% 40_70% etc and see the activity in different fractions. Do you mean that you did not have any activity of your protein till below 50% and the protein was present in fractions above 50%. The other possibility is that you prepared a cut from 0_50% and got activity in that fraction. Which is quite possible.
We use amm.sulfate to ppt proteins. If your protein is a hydrophobic it will get ppt . at lower con of salt. You try lower concentrations and conduct assay. Amm.sulfate is also toxic to proteins. Once you separate it from other proteins on a crude mixture you dialyze the ppt against water or dialute buffer. This way you can remove the salt
You can try different trials for your protein purification.
20, 40, 60, and 80 % ammonium sulphate saturación.
You separate, in each step, the precipitate by centrifugación, and quantify your interest protein in supernatant a precipitate, and calculate yield, specific activity, and purification factor (times than specífic activity increase).
According with results, you can adjust your trial to improve yield or purification factor, according your interest.
ie: 55, 78, and 85 saturation %
Yours,
Hector Nolasco
CIBNOR
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