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 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

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.

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


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


3. Resuspend the Dynabeads® in 250 µl 5 mM BS3.


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


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


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


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


8. 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.