6 After 15 min pour the contents from above step into a 50 mL centrifuge tube The tubes from all the groups have to be balanced by the TA before putting them in the centrifuge There is going to be addition removal of solutions All tubes will need to be sealed by the TA after balancing Centrifuge for 15 min at 9 000xg at 4 C to sediment the proteins quosdi A lu Tod anmuloo sii 7 After centrifugation you will see a pellet at the bottom of the tube and the liquid is called the supernatant a SUPERNATANT Pour off the supernatant into a beaker Transfer 1 mL into an Eppendorf tube label as S1 and put aside for protein electrophoresis Estimate the volume remaining in the beaker calculate the amount of ammonium sulfate you ll need to add to reach 50 saturation 85 g L of solution Add Ammonium sulfate slowly while stirring S1 on a stir plate Once the ammonium sulfate has been added continue to stir for 15 min dew IG ni blol 001 slamasz b PELLET Re suspend the pellet in 4 7 mL depending on the pellet size distilled water Gather dialysis tubing and clips from your TA Transfer the resuspended pellet into a dialysis bag and dialyze against Buffer A see recipe below Label this sample as P1 8 After S1 50 saturation supernatant has stirred for 15 min centrifuge S1 at 9 000xg 4 C for 15 min 13 9 Following centrifugation transfer 3 5 mL of the supernatant into a 15 mL conical tube label as S2 and put aside for protein electrophoresis Dissolve the pellet in 4 7 mL of distilled water use more water only if necessary Label this sample as P2 10 Gather dialysis tubing and clips from your TA Place the second pellet suspension into a separate dialysis bag and dialyze against Buffer A After dialysis P1 and P2 will be further purified based on differences in formal negative charge by ion exchange chromatography 11 At the next class transfer your dialyzed samples to falcon tubes