Preparing Immunoprecipitations for Immunoblotting.

Abstract

Immunoprecipitated proteins can be readily analyzed by immunoblotting. Proteins can be efficiently eluted from the Protein A or similar beads by addition of the SDS-PAGE sample loading buffer and heating at 95°C. This elution procedure will also remove the capturing antibody from the beads unless the antibody was cross-linked to the beads. Alternatively, the immunoprecipitated proteins as well as non-cross-linked capture antibodies can be eluted from the beads using low (2.1-2.8) or high (10-11) pH conditions. Incubation of the immunoprecipitates with the excess of the competing peptide allows the elution of the captured proteins without contamination of the sample with the antibodies present in the immunoprecipitates. However, this option is not always available, and the cost of competing peptide can be prohibitive for the routine immunoprecipitation/immunoblotting experiments. In this protocol, elution of the immunoprecipitated proteins from the beads is performed by mixing Protein A or similar beads containing the immunoprecipitated protein antigens of interest with SDS-PAGE sample buffer and boiling to prepare samples for protein gel electrophoresis.