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  • 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.
    Cross-linking Buffer: 0.2 M triethanolamine (pH 8.2)
    Blocking Buffer: 0.1 M ethanolamine (pH 8.2)
    Immunoglobulin in Binding Buffer

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

    Protocol

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

      Vortex and thoroughly resuspend Protein A Magnetic Beads.
    2. Aliquot 100 μl of bead suspension to a sterile microcentrifuge tube.
    3. Add 500 μl 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. Repeat wash.
    4. Add to the beads 80 μl of 0.1 M NaPhosphate Buffer (pH 8.0) and 15-25 μl of serum or 20 μg purified IgG in a maximum volume of 30 μl.
    5. Mix thoroughly and incubate at 4°C with agitation for 30 minutes.
    6. Apply magnet 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 beads (see cross-linking protocol) to create a reusable immunoprecipitation bead which prevents the co-elution of antibody with target protein.
    8. IgG Cross-linking to Protein A/G Magnetic Beads:
      Add 1 ml of Cross-linking Buffer (0.2 M triethanolamine, [pH 8.2]) to the Protein A/G immobilized antibody and vortex to resuspend. Apply magnet for 30 seconds, to pull beads to the side of the tube and remove supernatant. Repeat wash.
    9. 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.
    10. Apply magnet for 30 seconds, to pull beads to the side of the tube and remove supernatant.
    11. Add 1 ml Blocking Buffer (0.1 M ethanolamine, [pH 8.2]) and vortex to resuspend. Apply magnet for 30 seconds, to pull beads to the side of the tube and remove supernatant.
    12. Add 1 ml of Blocking Buffer and vortex to resuspend. Incubate for 1 hour at room temperature with agitation.
    13. Apply magnet for 30 seconds, to pull beads to the side of the tube and remove supernatant. Add 1 ml of PBS, vortex to resuspend, apply magnet for 30 seconds, to pull beads to the side of the tube and remove supernatant. Repeat wash twice.
    14. Add 1 ml Elution Buffer (0.1 M glycine-HCl [pH 2.5]) and vortex to resuspend, apply magnet for 30 seconds, to pull beads to the side of the tube and remove supernatant. This elutes bound antibody that is not cross-linked with DMP.
    15. Resuspend and store beads in 100 μl PBS, 0.1% Tween 20, 0.02% sodium azide.