Protein precipitation using ammonium sulfate current protocols. Addition of ammonium sulfate acidifies the solution so use at least a 50 mm hepes or tris buffer etc. Despite the potential demonstrated, nonviral gene therapy and dna vaccinations are still under development, and a therapeutic product or protocol has yet to. The basic theory of protein precipitation by addition of ammonium sulfate is presented. When high concentrations of small, highly charged ions such as ammonium sulfate are added, these groups compete with the proteins to bind to the water molecules. Ammonium sulfate precipitation procedure hyman lab. Infranatant obtained after centrifugation were taken in 3 ml centrifugation tubes and placed in a cooling jacket maintained at 0 while keeping the samples in ice jacket, 0.
Although other salts such as sodium sulfate are sometimes used, precipitation of antibodies is commonly done with ammonium sulfate. Also note that protein precipitation is not instantaneous. Salting out of proteins using ammonium sulfate precipitation article pdf available in methods in enzymology 541. While the sample is stirring, slowly add saturated ammonium sulfate to bring the final concentration to 50% saturation. Salt precipitation has been widely used to fractionate membrane proteins schagger, 1994. Optimization of isopropanol and ammonium sulfate precipitation. Optimisation of ammonium sulfate precipitation method to achieve.
Due to bound lipid andor detergents, ammonium sulfate precipitates have lower density than proteinonly precipitates. Large scale preparation and purification by ammonium. In solution, proteins form hydrogen bonds with water molecules through their exposed polar and ionic groups. The protein solution should be equilibrated to the temperature at which the precipitation procedure is carried. The addition of 40 45% ammounium sulfate precipitates igg which can be further purified by anion exchange chromatography. Thus, salting out can be used to separate proteins based on their solubility in the presence of a high concentration of salt. Unfortunately, it interferes with many procedures and tests such as immuno precipitation and mass spectrometry. At higher salt concentrations, protein solubility usually decreases, leading to precipitation. Protocol and % recovery introduction sodium dodecyl sulfate sds is an ionic detergent used in biological buffers to dissolve and denature proteins. Large volume precipitation of proteins with ammonium sulfate.
We conclude that carefully controlled ammonium sulfate precipitation and liquid chromatography. Add solid ammonium sulfate slowly with gentle stirring. Salting out of proteins using ammonium sulfate precipitation. Ammonium sulfate precipitation protocol exalpha biologicals inc. In this protocol, ammonium sulfate will be added incrementally to an e. It is very important to use fresh, desiccated ammonium sulfate.
The day before use, place ammonium sulfate over night in ca. Ammonium sulfate precipitation is one of the most commonly used methods for protein purification from a solution. Transfer sample to a beaker containing a stir bar and place on a magnetic stirrer. The cells are lysed using the mechanical force of agitation with glass beads and proteins are partially purified by an ammonium sulfate precipitation step. In this report, we evaluated the use of ammonium sulphate nh 4 2 so 4 precipitation. Protein precipitation using ammonium sulfate ncbi nih. Allow serum or ascitic fluid to thaw, determine total volume, and centrifuge at 3000g for 30 minutes. Ammonium sulfate precipitation combined with liquid. Pdf salting out of proteins using ammonium sulfate precipitation.