Antibody purification on protein A or protein G columns

From Diseasepedia

 

No1. Antibody purification on protein A or protein G columns

Antibody source	Protein A	Protein G
Monoclonal antibodies    mouse IgG1    Mouse IgG2a,IgG2b,IgG3    rat	V	V  V
Polyclonal antibody     human     rabbit     mouse    rat    horse    goat    donkey    pig    guinea pig    dog    cow	V  V    V    V  V  V  V  V	V  V  V

1.  Adjust the pH of the crude antibody preparation by adding 1/10 volume of 1.0 M Tris(pH8.0)

2.  Pass the antibody solution through a protein A or protein G bead column. These columns bind approximately 10-20 mg of antibody per milliliter of wet beads. Serum contains approximately 10mg/ml of total IgG, tissue culture supernatant contain 20-50 μg/ml of monoclonal antibody, and ascites between 1 and 10mg/ml.

     Note the approximate volume of the column bed because the wash and elution buffer are measured in relative values compared to the volume of beads being used.

3.  Wash the beads with 10 column volumes of 100mM Tris(pH8.0)

4.  Wash the beads with 10 column volumes of 100mM Tris(pH8.0)

5.  Elute the column with 50 mM glycine (pH3.0). Add this buffer stepwise as approximately 1/2 column volume per step. Collect the elute from each step in tubes containing 1/10 column volume of 1M Tris (pH8.0). Mix each tube gently to bring the pH back to neutral.

6.  Identify the immunnoglobulin-containing fractions by absorbance at 280nm. (1OD=approximately 0.8mg/ml)

 Combine the antibody-containing fractions and measure purity by running 2μg of total protein on a SDS-polyacrylamide gel and staining with Coomassie blue. Good purification should yield essentially pure heavy and light chain bands.