protocol_461.txt

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.