Antibody Purification ( small scale )
Centrifuge anti - sera @ 4ºC , 14,000g , 10min .
( Optional ) Filter through 0.2um filter .
Apply protein A or protein G beads gently to Bio - Rad empty gravity column .
Set up the column on a vacuum manifold .
Equilibrate column with 10 volumes of ice cold Binding Buffer .
Dilute anti - sera with 2 volumes of Binding Buffer .
Let diluted anti - sera go through the column by gravity .
Collect flow through and repeat step 6 once .
Wash column with 10 bed volumes of ice - cold Binding Buffer .
Elute antibody with 5 bed volumes of room temperature Elution Buffer .
Fill up each collecting tube with 200 μl 1M Tris - HCl ( PH9.0 ) .
Collect 1ml of flow through in each collecting tube .
Save for antibody activity test .
Re - equilibrate column with 10 bed volumes of Binding Buffer .
Store Column at refrigerator for later use .
