Eliminate antibody interference using the CLIP protocol with Dynabeads

Our cross-linking immunoprecipitation (CLIP) protocol combines UV cross-linking with Dynabeads immunoprecipitation. Downstream workflows includes mapping RNA protein binding sites to further understand post-transcriptional regulatory networks.

Co-elution of antibody heavy- and light-chain antibody fragments with the target antigen in immunoprecipitation (IP) procedures may interfere with downstream analysis. One way to eliminate this antibody interference is to covalently crosslink the immobilized antibody to the IP magnetic beads before performing the IP experiment. Because the antibody is chemically bound to the beads, the antibody remains attached to the beads upon elution of the antigen with nondenaturing, nonreducing elution buffers.

CLIP Sequence Protocol

The following protocol describes crosslinking of 5 µg IgG to 50 µL Dynabeads Protein A, Dynabeads Protein G, Immunoprecipitation Kit Protein A, or Immunoprecipitation Kit Protein G. The procedure uses Pierce BS3 Crosslinker (Cat. No. 21580), which is a water-soluble crosslinker that yields irreversible (stable amide) bonds between primary amines at physiological pH. This immunoprecipitation crosslinking protocol may be scaled up or down as required.

CLIP Protocol for IgG to Dynabeads Protein A or 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 µL 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 (starting from step 2.3 of the Immunoprecipitation protocol).

Buffers referenced in CLIP protocol

BS3 Conjugation Buffer: 20 mM Sodium Phosphate, 0.15M NaCl (pH 7-9)
BS3 Quenching Buffer: 1M Tris HCl (pH 7.5)