Search Thermo Fisher Scientific
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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.
The BS3 protocol should be the preferred/recommended protocol, when possible.
BS3 Conjugation Buffer: 20 mM Sodium Phosphate, 0.15 M NaCl (pH 7-9)
BS3 Quenching Buffer: 1 M Tris HCl (pH 7.5)
Crosslinking solution: Immediately before use, dissolve the DSS in dry DMSO at 10–25 mM. DSS is not soluble in water
Conjugation Buffer: Phosphate-buffered saline (PBS): 20 mM sodium phosphate, 0.15 M NaCl; pH 8. HEPES, bicarbonate/carbonate or borate buffers between pH 7 and 9 may be used as alternative buffers.
Quenching Buffer: 1 M Tris HCl (pH 7.5) (1 M glycine or lysine also may be used)
Note: The DSS crosslinker is moisture-sensitive. Allow to warm to room temperature before opening vials to prevent condensation formation. Store with a suitable desiccant. Dissolve DSS in DMSO or DMF immediately before use.
DSS is not compatible with amine-containing buffers and they should be avoided for the conjugation step (e.g., Tris, glycine).
For Research Use Only. Not for use in diagnostic procedures.