Immunoprecipitation Crosslinking

Cross-linking the immobilised antibody to the beads is often required to avoid co-elution of antibody heavy- and light chains with the target antigen when these may interfere with downstream analysis.

The following protocol describes cross-linking of 5 µg IgG to 50 µl  Dynabeads Protein A ,   Dynabeads Protein GImmunoprecipitation Kit Protein AImmunoprecipitation Kit Protein G  using the cross-linker BS3, which is a water-soluble crosslinker which yields irreversible cross-linking (stable amide bonds) at physiological pH. This immunoprecipitation cross-linking protocol may be scaled up or down as required.

Cross-linking protocol for IgGs immobilized to Dynabeads Protein A or Protein G

  1. Prepare 100 mM BS3 in Conjugation Buffer (Stock Solution). Proceed to making a 5 mM solution by diluting in Conjugation Buffer, 250 µl is required per sample.

  2. Note: BS3 Stock and Conjugation solutions must be freshly made prior to use!

  3. Wash the Ig-coupled Dynabeads Protein A or Protein G twice in 200 Conjugation Buffer. Place on magnet and discard supernatant.

  4. Resuspend the Dynabeads in 250 µl 5 mM BS3.

  5. Incubate at room temperature for 30 min with tilting/rotation.

  6. Quench the cross-linking reaction by adding 12.5 µl Quenching Buffer

  7. Incubate at room temperature for 15 min with tilting/rotation.

  8. Wash the cross-linked Dynabeads three times with 200 µl PBST (or IP buffer of your choice). Place on magnet and discard supernatant.

  9. Proceed with your IP and antigen elution.

BS3 Conjugation Buffer:
20 mM Sodium Phosphate, 0.15M NaCl (pH 7-9)

BS3 Quenching Buffer:
1M Tris HCl (pH 7.5)

Cross-linking reagent: Bis(sulfosuccinimidyl)suberate (BS3), f.ex. Cat. # 21580 from Thermo Fisher Scientific Inc.