Conjugation of magnetic beads for immunopurification of protein complexes

Abstract

Knowledge of the composition of protein complexes provides key insights into their functions. Immunoaffinity purification provides an effective means for isolating protein complexes and elucidating their composition. Immunoisolation is achieved with antibodies directed either specifically against the proteins of interest or against tags that are coupled to the proteins of interest. This approach uses immunoaffinity purification on magnetic beads coated with antibodies for the rapid and efficient purification of protein complexes from cells or tissues. This protocol describes conjugation of magnetic beads with antibodies in preparation for immunopreciptation of protein complexes.

FIGURE 1.

Binding capacity of magnetic affinity capture beads for antibodies. The graph shows that maximum binding is achieved with 7–8 μg of antibodies per mg of magnetic beads. (Reprinted, with permission, from the American Society for Biochemistry and Molecular Biology, Inc.)

FIGURE 2.

Using a magnetic concentrator to capture the magnetic beads for washing. The magnetic beads are held against the side of the tube by the magnetic particle concentrator while the liquid is carefully removed using a clean pipette tip.