Experiment 2 Purification of Y-globins from Human Serum Purpose 1.To master the principle and method of salting ou method in protein 2.To learn the principle and method of gel filtration in the extraction and purification of biological macromolecules. Principle The globins in human serum are classified into albumin,a-globins.B-globins and Y-globins.which can be separated by ammonium sulfate step-precipitation method depending on the difference of solubility.Pure Y-globins are collected afier removing salts from the fractions by gel filtration. Methods Step 1,Salting out (1).Add 1.0 ml serum into a centrifugal tube.and add 1.0 ml phosphate buffer saline (PB).Mix adequately.Then.add ml ammonium sulfate drop by drop,shaking the mixture constantly during the adding of ammonium sulfate After calming down at room temperature for 10-20 minutes.spin down the precipitation by centrifugation at 3,000 rpm for 10 min.Pour the supernatant into a clean tube (labeled A.which are mainly albumins).take the precipitation to(2). (2),Dissolve the precipitation in 1.0 ml PBS adequately in the centrifugal tube add 0.5 ml saturated ammonium sulfate drop by drop,remember keeping shaking the mixture.After calming down at room temperature for 10-20 minutes.spin down the precipitation by centrifugation at 3,000 rpm for 10 min.Pour the supernatant into a clean tube (labeled B.which are mainly a-and B-globins),the precipitation are primary purif fied -globins with salt.To get purer Y-globins,this step (2)can b repeated for 1-2 times. Step 2,Desalting (1)Colm packing.Fix the glass com(mn) shelf,keep it vertical and the bottom switch on.Stir up the Sephadex G-25 beads gently and pour about 10 ml into the column rapidly.When the column is filled,turn the bottom switch off.Knock the column gently by finger.Then turn the bottom switch on and let the liquid drop down slowly,about 8-10 drops per minute.When the