title: Western blotting via proximity ligation for high performance protein analysis.
abstract: Western blotting is a powerful and widely used method, but limitations in detection sensitivity and specificity, and dependence upon high quality antibodies to detect targeted proteins, are hurdles to overcome. The in situ proximity ligation assay, based on dual antibody recognition and powerful localized signal amplification, offers increased detection sensitivity and specificity, along with an ability to identify complex targets such as phosphorylated or interacting proteins. Here we have applied the in situ proximity ligation assay mechanism in Western blotting. This combination allowed the use of isothermal rolling circle amplification of DNA molecules formed in target-specific ligation reaction, for 16-fold or greater increase in detection sensitivity. The increased specificity because of dual antibody recognition ensured highly selective assays, detecting the specific band when combinations of two cross-reactive antitubulin antibodies were used (i.e. both producing distinct nonspecific bands in traditional Western blotting). We also demonstrated detection of phosphorylated platelet-derived growth factor receptor β by proximity ligation with one antibody directed against the receptor and another directed against the phosphorylated tyrosine residue. This avoided the need for stripping and re-probing the membrane or aligning two separate traditional blots. We demonstrate that the high-performance in situ proximity ligation-based Western blotting described herein is compatible with detection via enhanced chemiluminescence and fluorescence detection systems, and can thus be readily employed in any laboratory.
authors.affiliation: Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Rudbeck Laboratory, Uppsala University, SE-751 85 Uppsala, Sweden. yleen.liu@gmail.com
authors.forename: Yanling
authors.initials: Y
authors.lastname: Liu
authors.forename: Jijuan
authors.initials: J
authors.lastname: Gu
authors.forename: Åsa
authors.initials: Å
authors.lastname: Hagner-McWhirter
authors.forename: Poojahrau
authors.initials: P
authors.lastname: Sathiyanarayanan
authors.forename: Mats
authors.initials: M
authors.lastname: Gullberg
authors.forename: Ola
authors.initials: O
authors.lastname: Söderberg
authors.forename: Johan
authors.initials: J
authors.lastname: Johansson
authors.forename: Maria
authors.initials: M
authors.lastname: Hammond
authors.forename: Daniel
authors.initials: D
authors.lastname: Ivansson
authors.forename: Ulf
authors.initials: U
authors.lastname: Landegren
mesh_terms: D000906:Antibodies / Q000737:chemistry; D015153:Blotting, Western / Q000379:methods*; D002478:Cells, Cultured; D006801:Humans; D057230:Limit of Detection; D021141:Nucleic Acid Amplification Techniques; D009841:Oligonucleotides / Q000737:chemistry; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D020797:Receptor, Platelet-Derived Growth Factor beta / Q000378:metabolism; D059629:Signal-To-Noise Ratio; D014168:Transferrin / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism
chemical_list: D000906:Antibodies; D009841:Oligonucleotides; D014168:Transferrin; D014404:Tubulin; D020797:Receptor, Platelet-Derived Growth Factor beta