Cross-linking
Kirk Lab Protocol
Cross-linking protocol for IgGs immobilized to Dynabeads Protein G
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.
*Note: BS3 Stock and Conjugation solutions must be freshly made prior to use!
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) 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.
