Immunoprecipitation

      Principle

      Crosslink immunoprecipitation refers to the process of antigen purification utilising a specific antibody. The principle involves crosslinking to covalently attach a specific antibody to a protein A agarose resin (recombinant protein combining antibody-binding domains from protein A), which is then incubated with a protein mixture containing the antigen, allowing the antibody/antigen complex to form. After washing to remove unbound, undesired sample components, the antibody/antigen complex immobilised by crosslinking with disuccinmidyl suberate (DSS) on beaded agarose resin, is eluted and separated by centrifugation (see Figure 1). Samples under investigation were immunoprecipitated utilising a Pierce Crosslink IP kit, with all steps carried out at 4°C (unless otherwise stated) and all centrifugations at low speed (1000-3000 g) for 30-60 seconds.


      Fig. A schematic diagram of crosslink immunoprecipitation.

      Procedure

      Binding of antibody with Protein A Agarose Resin

      1. A solution containing 10 µg of antibody is prepared through dilution of the antibody with coupling buffer and ultrapure water.
      2. Then add 20 µl of Pierce Protein A/G Plus Agarose resin.
      3. Mix on a rotor for 30-60 minutes.
      4. Wash via centrifugation with coupling buffer.

      Crosslinking of bound antibody

      1. Resin complex is incubated with DSS, ultrapure water and coupling buffer and placed on a mixer for 30-60 minutes at room temperature.
      2. The solution is washed in Elution buffer and IP / Lysis Wash buffer via centrifugation, prior to proceeding to immunoprecipitation.

      Immunoprecipitation

      1. The sample under investigation is diluted with IP / Lysis Wash buffer to create a volume of 300-600 µl containing 500-1000 µg of total protein.
      2. The solution above is added to the prepared antibody resin complex and incubates overnight on a rotor at 4 °C.

      Antigen Elution

      1. The mixture is washed via centrifugation with IP / Lysis Wash buffer.
      2. Conditioning buffer and 5 ul of 1 M Tris (pH 9.5) are added to neutralize the low pH and allow functional assays.
      3. The sample is incubated and centrifuged with elution buffer and the eluate collected for further analysis or stored at -20 °C.