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.

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).

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)

1. Prepare 25 mM DSS in DMSO or DMF (Stock Solution). Proceed to making a 0.25 mM solution by diluting further in DMSO or DMF. Proceed to making a 5 mM solution by diluting in Conjugation Buffer, 250 µL is required per sample.
   Note: DSS Stock and Conjugation solutions must be freshly made prior to use! 
   
2. Wash the Ig-coupled Dynabeads Protein A or Protein G three times in 200 µL Conjugation Buffer. Place on magnet and discard supernatant.
3. Resuspend the Dynabeads in 250 µL of 1 to 5 mM DSS.
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).

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).