ROLE OF ATG POLYCLONAL ANTIBODIES(8)

PEG based Preparation of Plasma Proteins

Equal volumes of plasma and phosphate buffer are taken into a container and their pH is adjusted between 7.2 – 7.4. Keep it in refrigerator for 15-20 minutes. A calculated percentage of pre-chilled poly ethylene glycol is added with continuous mixing on magnetic stirrer. Mixing is done until PEG got dissolved and a visual precipitation is observed. After a continuous mixing this mixture is now kept in refrigerator for a good precipitation. Mixture is now transferred to centrifuge tubes and they are centrifuged at 5000 rpm for 6 months. Now the pellet is separated and supernatant is stored for the comparative study in the future proceedings. Pellet is now taken to the dialysis beaker where they dialyzed against phosphate buffer.

SDS PAGE

SDS is a reagent that readily binds to proteins. At pH 7.0 in the presence of 1 % w/v SDS and 2-mercaptoethanol, proteins dissociate in to their subunits and bind large quantities of detergents. Under these conditions most of the proteins bind about 1.4g of SDS/gm of protein, which in turn completely masks the natural charge of the proteins and gives a constant charge to mass ratio. Larger the molecule, greater will be the charge. Hence the electrophoretic mobility of the complex depends upon the size (molecular weight) of the protein, where as the plot of log molecule weight against relative mobility gives a straight line. In the experiment, the molecular weight of protein is determined by comparing its mobility with a series of protein standards.