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1. (WO2017040404) A METHOD OF DETECTING MOLECULES IN PROXIMITY TO A TARGET MOLECULE IN A SAMPLE

Pub. No.:    WO/2017/040404    International Application No.:    PCT/US2016/049263
Publication Date: Fri Mar 10 00:59:59 CET 2017 International Filing Date: Tue Aug 30 01:59:59 CEST 2016
IPC: G01N 33/68
Applicants: THE UNITED STATES OF AMERICA AS REPRESENTED BY THE SECRETARY OF THE DEPARTMENT OF HEALTH AND HUMAN SERVICES
Inventors: BAR, Daniel Z.
COLLINS, Francis
Title: A METHOD OF DETECTING MOLECULES IN PROXIMITY TO A TARGET MOLECULE IN A SAMPLE
Abstract:
Antibody-based methods of detecting of molecules interacting with a target molecule, such as a nuclear or cytoplasmic protein, in a fixed and, optionally, permeabilized cell or tissue sample. In one example, the target molecule is bound by a primary antibody, and a secondary, peroxidase-conjugated antibody, then binds to the primary antibody. The peroxidase may also be conjugated the primary antibody. Biotin tyramide is added to the sample, and the peroxidase generates short-lived intermediates resulting in biotinylation of molecules adjacent to the peroxidase. Biotinylated molecules can be isolated from the sample by affinity interaction with avidin- or streptavidin, resulting in a fraction containing biotinylated molecules that are located in the sample in proximity to the target molecule. The fraction can be analyzed by mass-spectroscopy, Western blotting, sequencing and other techniques.