Immunoprecipitation protocol

Immunoprecipitation Protocol Immunoprecipitation (IP) is one of the most widely used antibody-based techniques.

It is used to purify and enrich the protein of interest from a complex mixture such as cell lysate, tissue homogenate or blood sample. The protein is captured by a specific antibody, and then the antibody-protein complexes are pulled out of the sample using Protein A/G-coupled agarose beads or magnetic beads. IP is an important step in many proteomics studies: the molecular weights of protein antigens, protein/protein interactions, post-translational modifications and expression profiling of proteins. The IP technique can also enable the detection of rare proteins which otherwise would be difficult to detect since they can be concentrated up to 10,000-fold by immunoprecipitation. Although researchers are increasingly choosing magnetic beads for IP purification, agarose-based IP remains a versatile and powerful option in many circumstances, especially where the need for scale-up is anticipated.

Here we describe a detailed procedure for agarose-based IP to purify and enrich the protein of interest.

Reagents: PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH to 7.4.

Cell lysis buffer: 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1mM EDTA, 1% NP-40, 1% Na-deoxycholate, 0.1% SDS, sterile-filtered, add protease inhibitor cocktail before use.

3 × SDS buffer: 150mM Tris-HCl (pH6.8), 6%(W/V) SDS, 0.3%(W/V) BPB, 30% glycerol, 3% β-mercaptoethanol.

Glycine buffer: 0.10 M Glycine, 500 mM NaCl, 0.05M Tris-HCl (pH 1.5–2.5) Tris-HCl: 1 M Tris, 4% (W/V) HCl, pH 8.0 Antibody https://www.creative-diagnostics.com/immunoprecipitation-ip-protocol.htm