Protein Precipitation Mehod

(1) Clarify the protein solution (in most cases the lysates) by centrifugation.
(2) Transfer the supernatant into an ice cold beaker with a magnetic bead.
(3) Note the exact amount of the supernatant.
(4) Keep the beaker chilled by placing it in an ice tray.
(5) Transfer the beaker with the ice tray onto a magnetic stirrer

(6) Weigh the amount of ammonium sulfate to be added. The amount depends on the volume of the solution and the percentage saturation of the salt needed. Refer to the precipitation chart. In case of protein purification, a step precipitation is carried out.
(7) Slowly add the ammonium sulphate with stirring. One needs to be careful as the addition of the salt should be very slow. Add a small amount at a time and then allow it to dissolve before further addition.
(8) Keep it on the stirrer for 1hr precipitation to occur in ice.
(9) Centrifuge at 10,000g for 15min at 4oC.
(10) The pellet contains the precipitated protein which could be dissolved in a suitable buffer for further analysis and purification.
(11) For a second round of precipitation of a different protein, the supernatant is again used and the above same steps are followed.

Protein Precipitation

Many cytosolic proteins are water soluble and their solubility is a function of the ionic strength and pH of the solution. The commonly used salt for this purpose is Ammonium Sulphate, due to its high solubility even at lower temperatures. Proteins in aqueous solutions are heavily hydrated, and with the addition of salt, the water molecules become more attracted to the salt than to the protein due to the higher charge. This competition for hydration is usually more favorable towards the salt, which leads to interaction between the proteins, resulting in aggregation and finally precipitation. The precipitate can then be collected by centrifugation and the protein pellet is re-dissolved in a low salt buffer. Since different proteins have distinct characteristics, it is often the case that they precipitate (or ‘salt out’) at a particular concentration of salt.