Home Protocols Cross-linking of IgG to Protein A or G Beads

Cross-linking of IgG to Protein A or G Beads

Overview

Materials Needed:

Protein A (NEB #S1425S) or Protein G (NEB #S1430S) Magnetic Beads
Elution Buffer: 0.1 M glycine-HCl (pH 2.5)
Binding Buffer: 0.1 M NaPhosphate Buffer (pH 8.0)
Dimethyl pimelidate dihydrochloride (Sigma, D-8388) dissolved at 25 mM in Cross-linking Buffer. Note: DMP is unstable in aqueous solution. Prepare solution immediately prior to use
Cross-linking Buffer: 0.2 M triethanolamine (pH 8.2)
Blocking Buffer: 0.1 M ethanolamine (pH 8.2)
10x PBS (Thermo, 70011044)
Tween 20 (Sigma Aldrich, P9416-50ML)
Sodium azide (Sigma Aldrich, 71289)
Immunoglobulin in Binding Buffer

This protocol consists of an IgG immobilization step followed by covalent cross-linking of the IgG to the Protein A/G solid support. For IgG that has been previously immobilized on the beads, proceed directly to the cross-linking protocol.

Protocol

IgG Immobilization:

The following protocol is for the binding of 20 μg of purified IgG or isolation of 20 μg IgG from serum.

  1. Vortex and thoroughly resuspend Protein A or Protein G Magnetic Beads.
  2. Aliquot 100 μl of bead suspension to a sterile microcentrifuge tube.
  3. Add 500 μl Binding Buffer (0.1 M NaPhosphate Buffer, pH 8.0) and vortex to resuspend. Apply magnet for 30 seconds to pull beads to the side of the tube and remove supernatant.
  4. Repeat step 3.
  5. Add to the beads 80 μl of Binding Buffer (0.1 M NaPhosphate Buffer, pH 8.0) and 20 μg purified IgG in a maximum volume of 30 μl (alternatively, add 15-25 μl of serum containing IgG of interest).
  6. Mix thoroughly and incubate at 4°C with agitation for 30 minutes.
  7. Apply magnet and remove supernatant.
  8. Wash beads three times as in step 3.
  9. Add 1 ml of Cross-linking Buffer (0.2 M triethanolamine, pH 8.2) to the beads and gently vortex to resuspend. Apply magnet for 30 seconds to pull beads to the side of the tube and remove supernatant.
  10. Repeat step 9.
  11. Resuspend in 1 ml Cross-linking Buffer containing 25 mM DMP (6.5 mg DMP/ml of buffer). Mix thoroughly and incubate at room temperature for 45 minutes with agitation.
  12. Apply magnet for 30 seconds to pull beads to the side of the tube and remove supernatant.
  13. Add 1 ml Blocking Buffer (0.1 M ethanolamine, pH 8.2) and gently vortex to resuspend. Apply magnet for 30 seconds to pull beads to the side of the tube and remove supernatant.
  14. Add 1 ml of Blocking Buffer and vortex to resuspend. Incubate for 30 minutes at room temperature with agitation.
  15. Apply magnet for 30 seconds to pull beads to the side of the tube and remove supernatant.
  16. Add 1 ml of PBS, gently vortex to resuspend, apply magnet for 30 seconds to pull beads to the side of the tube and remove supernatant.
  17. Repeat step 16 twice.
  18. Add 1 ml Elution Buffer (0.1 M glycine-HCl, pH 2.5) and gently vortex to resuspend, apply magnet for 30 seconds to pull beads to the side of the tube and remove supernatant.
    19.  This elutes bound antibody that is not cross-linked with DMP.
  19. Add 1 ml of PBS, gently vortex to resuspend, apply magnet for 30 seconds to pull beads to the side of the tube and remove supernatant.
  20. Repeat step 19 twice.
  21. Resuspend and store beads in 100 μl PBS, 0.1% Tween 20, 0.05% sodium azide.