title: Immunoblotting and immunodetection.
abstract: Immunoblotting (western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. This unit provides protocols for all steps, starting with solubilization of the protein samples, usually by means of SDS and reducing agents. Following solubilization, the material is separated by SDS-PAGE and the antigens are electrophoretically transferred to a membrane, a process that can be monitored by reversible staining with Ponceau S. The transferred proteins are bound to the surface of the membrane, providing access to immunodetection reagents. After nonspecific binding sites are blocked, the membrane is probed with the primary antibody and washed. The antibody-antigen complexes are tagged with horseradish peroxidase or alkaline phosphatase coupled to a secondary anti-IgG antibody, and detected using appropriate chromogenic or luminescent substrates. Finally, membranes may be stripped and reprobed.
authors.affiliation: UVP, Inc., Upland, California, USA.
authors.forename: Sean
authors.initials: S
authors.lastname: Gallagher
authors.forename: Scott E
authors.initials: SE
authors.lastname: Winston
authors.forename: Steven A
authors.initials: SA
authors.lastname: Fuller
authors.forename: John G R
authors.initials: JG
authors.lastname: Hurrell
mesh_terms: D000906:Antibodies / Q000032:analysis; D000936:Antigen-Antibody Complex / Q000032:analysis; D019921:Biotinylation; D015153:Blotting, Western; D002863:Chromogenic Compounds / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting / Q000379:methods*; D049408:Luminescent Agents / Q000032:analysis; D013194:Staining and Labeling
chemical_list: D000906:Antibodies; D000936:Antigen-Antibody Complex; D002863:Chromogenic Compounds; D049408:Luminescent Agents