My protein is 118 kDa. 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?
You have to start first from less ammonium sulphate concentration (say 40%) to the higher. This is performed by adding increasing amounts of ammonium sulphate and collecting the different fractions of precipitate protein. You have to test it. Some proteins may require very higher concentrations to precipitate. Separation of proteins by ammonium sulfate gradient solubilization method helps you to get a fraction that mainly consists of your protein. Dyalyse each fraction and see which fraction contains your protein mostly and then you can go further to chromatographic step.
If you can test for activity of your protein, you could start with a broad range cut, say 40 and 80% and find which fraction activity is retained in. From there you can fine tune the saturation in 10% gradients if you wanted to establish a more refined cut. Temperature stability is essential for all steps. Dialysis membranes are an effective way of removing the ammonium sulphate for downstream chromatography or alternatively desalting or buffer exchange/concentrator columns can be used such as the PD10's or vivaspin. It may also be worth considering affinity chromatography targeted to your protein of interest due to its high specifity as an onwards step.
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