Antibodies purification from serum or ascite

IgG from the most species binds Protein A Sepharose CL-4B at neutral pH and physiological ionic strenght.

Available binding capacity (there might be considerable deviation in binding capacity for different immonoglobulins): 20 mg human IgG/ml drained beads

Dilute serum (max 10 mg/ml IgG) 1:10 with 20mM Sodium Phosphate, pH:7,0 and add PASepharose or PGAgarose (0,5 ml drained beads / 10ml diluted serum ).

O.N. rotanting 4°C

Elution: 0.1M glycine buffer pH:2,5 (or 0.1M citric acid buffer). Add glycine same vol of drained beads and centrifuge 15 sec 6000 rpm in eppendorf. As a safety measure to preserve the activity of acid labile IgGs it is recommended to add 200ul 1M Tris-HCl, pH=8.0 per ml to neutralize the eluted fraction.

Dialize against PBS on 4°C then check OD 280 and store (4°C adding azide or 50% glycerol).