[박상규] Purification of IgG from serum

[Anonymous]

Purification of IgG from serum (1ml)

 

1.     Dilute serum with 1XPBS 1:1

2.     Equilibrate the protein A agarose (rabbit serum) or protein G agarose (mouse ascites) (600ul) with 1ml of 1X PBS, 3 times

3.     Load the diluted serum into pre-equilibrated protein A agarose

4.     Harvest the pass-through (DO NOT discard). Reload the pass-through into the protein A agarose column repeat 2 times.

5.     Wash the column with 1ml of 1X PBS 3 times.

6.     Add 100mM Glycine(pH 2.5) 600ul and harvest pass-through in eppendorf tube filled with 60ul 1 M Tris-HCl (pH 8.0)and invert the tube to fully mix the solution.

7.     Repeat 5 times step 5

8.     Quantify the IgG with Bradford assay

9.     Pick up the fraction and exchange the buffer with 1X PBS containing 15% glycerol using PD10 column.

10.  Quantify protein and store at –70 degree.

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