FLIP (FLUORESCENCE IMMUNOPRECIPITATION) FOR HIGH-THROUGHPUT IMMUNOPRECIPITATION

摘要

This application describes an assay for immunoprecipitation that is quick, reliable, easy to perform, and that can be used in a high throughput fashion because it does not rely on western blotting analysis even if it can be included in a standard IP/WB procedure without affecting the output of the analysis. Because of these features the FLIP assay is ideal for the high-throughput screening of IP-grade antibodies. Here we present the basic concept of the invention and the application of the FLIP in high-throughput screening such as the quick identification of IP-proficient mouse monoclonal antibodies.

主权项

1. A method for the identification of antibodies able to recognize a target protein in its folded state, comprising:
expressing a target protein operatively linked to a fluorescent protein in a host cell; collecting a crude or partially purified host cell lysate; mixing said lysate with a primary antibody that binds to said target protein and beads coated with an affinity reagent, wherein the affinity reagent binds to the primary antibody, creating a lysate-bead mixture that comprises the primary antibody bound to the target protein and the bead coated with the affinity reagent; centrifuging said lysate-bead mixture; collecting the lysate-bead mixture; and measuring fluorescence of the lysate-bead mixture using a manual fluorescence microscope, an automated microscopy system, or a fluorimeter.