PROXIMITY LIGATION TECHNOLOGY FOR WESTERN BLOT APPLICATIONS

摘要

The invention provides a method for detecting a biomolecular feature (a protein, protein complex, or modified protein such as a phosphorylated protein) by a modified Western blot type of assay, which method either electrophoretic gel separation followed by transfer, or direct spotting of a sample containing the biomolecular feature onto a membrane; providing a proximity probe pair, each probe comprising a binding moiety with affinity for a different binding site on the bio molecular feature and a reactive oligonucleotide, coupled thereto; binding the proximity probes to their respective binding sites on the biomolecular feature through the binding moiety, adding a splint oligonucleotide and a backbone oligonucleotide which are complementary to the reactive oligonucleotide pair, and allowing hybridization among them; ligating the hybridized DNA oligonucleotides to create a circularized DNA molecule when both probes bind sufficiently close to each other on the bio molecular feature, amplifying the circularized DNA by isothermal amplification; and detecting the presence and quantity of the bio molecular feature using a detection oligonucleotide complementary to the amplification product. Also provided are methods for multiplexed detection of more than one bio molecular feature, as well as kits for performing the assays.