Protocol

Immunoprecipitation: Purifying the Immune Complexes

This protocol was adapted from "Immunoprecipitation," Chapter 7, in Using Antibodies by Ed Harlow and David Lane. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 1999.

INTRODUCTION

In this protocol, immune complexes are formed by the addition of antibodies to cell lysates. The antibodies bind to their cognate antigen from a lysate. Protein A or protein G beads are then added to the solution containing antibody-antigen complexes, and the proteins that do not bind to the beads are removed by washing. The correct controls for immunoprecipitation should include nonimmune antibodies that are as close as possible to the specific antibody; for example, polyclonal serum should be compared to polyclonal serum from the same species (ideally a prebleed from the same animal used for immunization). For monoclonal antibodies, the control must be from the same source as the specific antibody. Do not use tissue culture supernatants from parental myeloma cells as they do not contain antibodies. Suitable control hybridoma cell lines can be obtained from the American Type Culture Collection and the European Collection of animal cell cultures.