Crosslinking Immunoprecipitation Protocol using 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.
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.
BS3 Conjugation Buffer: 20 mM Sodium Phosphate, 0.15M NaCl (pH 7-9)
BS3 Quenching Buffer: 1M Tris HCl (pH 7.5)
For Research Use Only. Not for use in diagnostic procedures.