[박상규] Purification of IgG from serum

[Anonymous]

Purification of IgG from serum

 

1.     Add 1ml of serum to 10ml of IP buffer

2.     Add 200ul protein A(mouse serum : protein G, 50ul) agarose to serum/ IP buffer mixture(conical tube)

3.     rotate for 2 hrs at 4 degree

4.     spin down at 800Xg for 5 min

5.     discard the sup carefully by using long-tip suction master.

6.     Add 5ml IP buffer and resuspend the pellet.

7.     rotate for 5min and spin down as step 3, 4

8.     repeat step 7 two times

9.     resuspend the pellet with 1ml of IP Buffer

10.  transfer the solution to open column carefully not to lose the beads.

11.  pass the buffer and add 100mM Glycine(pH 2.5) 300ul and harvest the pass through in the 110ul of 1M Tris-HCl(pH 8.0) filled eppendorf tube.

12.  add 100mM Glycine(pH 2.5) 800ul and harvest pass through in step 11 eppendorf tube and invert the tube to fully mix the solution.

13.  transfer the IgG solution(1ml) to 6ml viva spin(cut off : 50,000) and add 4ml 1X PBS containing 20% glycerol and mix well.

14.  Centrifuge the viva spin at 2,500Xg for 15 min and, up and down the solution by using pippete not to aggregate the IgG around viva spin membrane.

15.  repeat step 14 till the solution will be 1ml

16.  Add 4ml 1X PBS containing 20% glycerol and mix well and repeat step 14, 15 four tmes.

17.  Quantify the protein and store at –70 degree.

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