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 G, Immunoprecipitation Kit Protein A , immunoprecipitation 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
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.
Note: BS3 Stock and Conjugation solutions must be freshly made prior to use!
- Wash the Ig-coupled Dynabeads Protein A or Protein G twice in 200 µl Conjugation Buffer. Place on magnet and discard supernatant.
- Resuspend the Dynabeads in 250 µl 5 mM BS3.
- Incubate at room temperature for 30 min with tilting/rotation.
- Quench the cross-linking reaction by adding 12.5 µl Quenching Buffer
- Incubate at room temperature for 15 min with tilting/rotation.
- Wash the cross-linked Dynabeads three times with 200 µl PBST (or IP buffer of your choice). Place on magnet and discard supernatant.
- Proceed with your IP and antigen elution (starting from step 2.3 of the Immunoprecipitation protocol).
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.