Protocol created by Lorena Maestre - Monoclonal Antibodies Unit, Centro Nacional de Investigaciones Oncológicas
This technique consist on the isolation of an antigen present in a complex sample through the precipitation with its specific antibody. Using this technique any antigen present in one cell or tissue extract can be purified in order to be visualized in an acrylamide gel. One important factor is the antibody affinity against the antigen, antibodies with affinities levels lower than 107 mol-1 are very difficult to be used in this technique. In the same way it is very important to know the immunoglobulin isotype of our antibody in order to select the suitable protein-agarose for the immunoprecipitation.
- Protein G PLUS-Agarose (Santa Cruz sc-2002)
- Protein A/G PLUS-Agarose (Santa Cruz sc-2003)
- HNTG buffer (see Appendix I)
- Protease inhibitor cocktail tablets (Roche 05 056 489 001)
- Prepare the immunoprecipitation mix (300 μg cellular or tisular extract/300 μl extraction buffer/immunoprecipitation).
- Add the antibody used for the immunoprecipitation (2μl in case of commercially or purified antibodies and 40μl in case of supernatants).
- Keep 2 hours to overnight at 4º with rotation.
- Add 20 μl of Protein G PLUS-Agarose or Protein A/G PLUS-Agarose (it can be blocked previously with dry milk powder)
- Keep in a tube rotator at 4º for 30 minutes.
- Centrifuge at 13000rpm for 1 min, discard the supernatant and add 1 ml of HNTG buffer (Appendix I). Mix gently and centrifuge again. Repeat this step 4 times, use PBS for the last wash.
- Discard as much supernatant as possible and add 15 μl of 2X loading buffer (Appendix I).
- Heat the samples for 5 min at 95º, centrifuge and load into the gel.
- Continue with the Western Blotting protocol. If it is possible, use for the Western Blotting technique one primary antibody different (produced in other species or against other antigen of the same protein) from the one used in the immunoprecipitation.
- 20mM Hepes pH7.5
- 150mM NaCl
- 0.1% Triton X-100
- 10% Glycerol 10%
Extraction buffer (prepare just before use)
- 500μl Lysis buffer
- 40μl protease inhibitors
- 460μl distilled water
2x Lysis buffer
- 50mM Tris-HCl pH 7.5
- 150mM NaCl
- 1% Igepal
2X Loading buffer
- 100mM TrisHCl pH 6.8
- 4% SDS
- 0.2% Bromophenol blue
- 20% Glycerin
- 10% Mercaptoethanol