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.
Highlights
From the ‡Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Rudbeck Laboratory, Uppsala University, SE-751 85 Uppsala, Sweden; §GE Healthcare Bio-sciences AB, Rapsgatan 23, SE-751 84 Uppsala, Sweden; ¶Olink AB, Dag Hammarskjoldsvag 54A, SE-751 83 Uppsala, Sweden
Signal amplification is possible by e.g. real-time PCR for detection of proteins in solution (9 –12), or by an isothermal rolling circle amplification (RCA) of circularized reporter DNA strands for localized detection of target proteins in cells and tissue sections by in situ proximity ligation assay (PLA) (Fig. 1) (13)
Western Blotting via Proximity Ligation by no more than a few tens of nanometers allows hybridization of two oligonucleotides, which can be ligated into a circle, in reactions templated by the oligonucleotides attached to the antibodies
Summary
Yanling Liu‡§ʈ, Jijuan Gu‡, Åsa Hagner-McWhirter§, Poojahrau Sathiyanarayanan‡, Mats Gullberg¶, Ola Soderberg‡, Johan Johansson§, Maria Hammond‡, Daniel Ivansson§, and Ulf Landegren‡. 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. 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. Received May 12, 2011, and in revised form, July 1, 2011 Published, MCP Papers in Press, August 2, 2011, DOI 10.1074/ mcp.O111.011031 1 The abbreviations used are: WB, Western blotting; ECL, enhanced chemiluminescence; PLA, proximity ligation assay; RCA, rolling circle amplification; DMEM, Dulbecco’s Modified Eagle Medium; FCS, fetal calf serum; PDGF, platelet-derived growth factor; PDGFR, ular biological laboratories since it was first published in 1981 (1). Binding by the oligonucleotide-modified antibodies to pairs of epitopes separated platelet-derived growth factor receptor ; RT, room temperature; HRP, horse radish peroxidase
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