Highly sensitive and specific protein detection via proximity ligation in capillary westerns
(English)Manuscript (preprint) (Other academic)
Detection and quantification of proteins and their post-translational modifications are crucial todecipher functions of complex networks in cell biology and medicine. Affinity-based protein analyseshave an important role in proteomic research, and western blotting is one of the most-widely usedtechniques. The NanoPro 1000 system developed by ProteinSimple is an instrument that can resolveand identify proteins and their isoforms through capillary isoelectric focusing with antibody baseddetection directly in the capillaries, in analogy to conventional western blotting. The in situ proximityligation assay (PLA) serves to detect the location of proteins using pairs of oligonucleotide-conjugatedantibodies that mediate formation of rolling circle amplification (RCA) products upon recognition ofthe target protein, or single or dual primary antibodies binding the target. PLA can offer improvedspecificity of detection, and increased sensitivity via, RCA initiated by oligonucleotides attached tothe antibody pairs. Here we have used in situ PLA to detect single or pairs of primary antibodiesbinding proteins separated in the NanoPro 1000 system for highly sensitive and specific detection ofproteins and their isoforms. We achieved at least 10-fold improved detection limits for Erk protein inHDMEC cell lysates, compared with the standard NanoPro 1000 assays, and we demonstrated greatlyenhanced specificity of detection by combining pairs of antibodies, each of which exhibited crossreactivesignals when used on their own.
Biochemistry and Molecular Biology
IdentifiersURN: urn:nbn:se:uu:diva-236588OAI: oai:DiVA.org:uu-236588DiVA: diva2:764718