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 with 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 or Ponceau S staining. 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: D000818:Animals; D000937:Antigen-Antibody Reactions; D001360:Avidin; D001391:Azo Compounds; D019921:Biotinylation; D015153:Blotting, Western / Q000295:instrumentation / Q000379:methods*; D002863:Chromogenic Compounds / Q000032:analysis; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D007124:Immunoenzyme Techniques; D007202:Indicators and Reagents; D008163:Luminescent Measurements; D008567:Membranes, Artificial; D011506:Proteins / Q000032:analysis* / Q000276:immunology; D012394:Rosaniline Dyes; D012967:Sodium Dodecyl Sulfate; D013194:Staining and Labeling / Q000379:methods
chemical_list: D001391:Azo Compounds; D002863:Chromogenic Compounds; D007202:Indicators and Reagents; D008567:Membranes, Artificial; D011506:Proteins; D012394:Rosaniline Dyes; D001360:Avidin; D012967:Sodium Dodecyl Sulfate; C048139:Coomassie blue; C032756:ponceau S