Purification of IgG using Protein A/G Magnetic Beads

Introduction

The following protocol is for the isolation of 10 µg of IgG from approximately 20 µl of human serum.

Buffer

Wash/Binding Buffer

Protocol

1. Isolation
   
   The following protocol is for the isolation of 10 µg of IgG from approximately 20 µl of human serum.
   
   (1) Vortex and thoroughly resuspend Protein A/G Magnetic Beads.
   (2) Aliquot 25 μl of bead suspension to a sterile microcentrifuge tube.
   (3) Add 500μl of Binding Buffer (0.1 M sodium phosphate, pH 8.0) and vortex to resuspend. Apply magnetic field for 30 seconds to pull beads to the side of the tube (Magnetic Separation Rack [NEB #S1506 ] holds 6 microcentrifuge tubes). Remove supernatant. Repeat wash.
   (4) Add 80 μl of Binding Buffer and 15?25 μl of serum to the beads.
   (5) Mix thoroughly and incubate at 4°C for 30 minutes with agitation.
   (6) Apply magnetic field and remove supernatant.
   (7) Wash beads three times as in step 3.
   
   At this point the purified IgG can be eluted from the beads or used directly for immunoprecipitation of target proteins. The purified IgG can also be cross-linked to the Protein A/G Magnetic Beads to create a reusable immunoprecipitation bead which prevents the co-elution of antibody with target protein (2,3).
   
   
2. IgG Elution
   
   (1) Add 50 μl of 0.2 M glycine (pH 2.5) to the bead pellet, vortex and incubate for 5 minutes at 4°C with agitation.
   (2) Apply magnetic field and transfer eluted IgG to clean microcentrifuge tube.
   (3) Add an additional 50 μl of 0.2 M glycine to the beads and repeat elution step. Pool elution supernatants and neutralize by adding 20 μl of 1 M Tris-HCl (pH 9.0). Store at 4°C.