2011-2012 Catalogue - PeproTech, Inc.

www.peprotech.com2011-2012 CatalogueWestern Transfer (Western Blot)INFORMATION & TECHNICAL SUPPORTWestern Transfer, also known as Western Blotting,is a rapid immunoblotting technique for identifyingthe presence of a particular protein in a complexmixture of proteins such as cell lysates or sera. Thetechnique exploits both the efficiency of SDS-PAGE toseparate a mixture of proteins into distinct protein bands,and the ability of immunochemical reagents to interactspecifically with a given protein antigen.A typical Western-transfer protocol (e.g. for thedetection of protein X) includes the following steps:(1) The proteins in the tested solution are separated intodistinct bands by SDS-PAGE, (2) The size-separatedproteins are transferred from the polyacrylamide gel toa nitrocellulose membrane (or another suitable matrix).(3) The membrane containing the protein bands is seriallyincubated with: (a) a suitable blocking reagent toprevent non-specific protein binding, (b) a wash solutionto rinse any unbound blocking reagent, (c) a probingantibody (anti-protein-X antibody) that forms a specificimmune complex with Protein-X, (d) additionalwash solution to remove any unbound antibody, (e) anenzyme-linked antibody that binds specifically to theF cregion of the anti-Protein-X antibody, (f) additionalwash solution to remove any unbound enzyme-linkedantibody, and finally, (g) a substrate solution, which inthe presence of the enzyme, yields an insoluble, coloredproduct that precipitates at the site of the immunecomplex, thereby rendering the Protein X band visible(Figure III-1).Typical Western Transfer andDevelopment Protocol• *Western Transfer Buffer (1/2 x TOWBIN)Tris Base..............1.45g (12mM finalconcentration)Glycine................7.2g (96mM finalconcentration)Methanol.............200mL (20% finalvolume)diH 2O...................add to 1.0L final volume• Antigen specific probing antibody (PrimaryAntibody)• Secondary antibody (Donkey-anti-probingantibody species conjugated to AlkalinePhosphatase)• Commercially available alkaline phosphataseconjugate substrate kitTransfer Protocol1. Load the protein sample onto a 4-20% Tris-Glycine polyacrylamide gel and run until desiredresolution is achieved. (The electrophoresis can befollowed by using pre-stained molecular weightmarkers).Continued…Figure III-1 - Schematic presentation ofWestern Transfer Protocol-+Materials Needed• SDS-PAGE apparatus and accessories• Submerged Western Transfer Cassette andaccessories• A nitrocellulose membrane, approximatelythe size of the gel, presoaked in WesternTransfer Buffer* for five minutes. (Note:The membrane should be handled withgloves and clean forceps to avoidcontamination with extraneous proteins.)164To Order: EC: Tel: +44 (0)20 7610 3062 • email: info@peprotech.co.ukBestellungen: AUSTRIA: Tel: +43 (0)1 405 9696 • email: info@peprotech.atObjednávky: Česká Rep.: Tel: +420 225 992 284 • email: info@peprotech.cz