title: A streamlined Western blot exercise: An efficient and greener approach in the laboratory classroom.
abstract: SDS-PAGE and western blotting are two commonly taught protein detection techniques in biochemistry and molecular biology laboratory classrooms. A pitfall associated with incorporating these techniques into the laboratory is the significant wait times that do not allow students to obtain timely results. The waiting associated with SDS-PAGE comes from staining and destaining, whereas with western blotting it is the times required for antibody incubations and the numerous wash steps. This laboratory exercise incorporates 2,2,2-trichloroethanol (TCE) into the SDS-PAGE gel allowing for visualization of migrated proteins in a matter of minutes, saving both the time and chemical waste associated with traditional Coomassie staining. Additionally, TCE staining does not affect protein transfer eliminating the requirement for duplicated gels for total protein and western analyses. Protein transfer can be confirmed immediately without the use of Ponceau S staining. Lastly, this western blot procedure has been further shortened by using an HRP-conjugated primary antibody, which eliminates the secondary antibody incubation and washes, and uses a colorimetric detection to allow for visualization by students without the need for specialized equipment.
authors.affiliation: Department of Biology, Armstrong State University, Savannah, Georgia, 31419.
authors.forename: Traci L
authors.initials: TL
authors.lastname: Ness
authors.affiliation: Department of Biology, Armstrong State University, Savannah, Georgia, 31419.
authors.forename: Rebekah L
authors.initials: RL
authors.lastname: Robinson
authors.affiliation: Department of Chemistry and Physics, Armstrong State University, Savannah, Georgia, 31419.
authors.forename: Wais
authors.initials: W
authors.lastname: Mojadedi
authors.affiliation: Department of Biology, Armstrong State University, Savannah, Georgia, 31419.
authors.forename: Lydia
authors.initials: L
authors.lastname: Peavy
authors.affiliation: Department of Chemistry and Physics, Armstrong State University, Savannah, Georgia, 31419.
authors.forename: Mitch H
authors.initials: MH
authors.lastname: Weiland
mesh_terms: D015153:Blotting, Western / Q000379:methods*; D003479:Curriculum; D004493:Education / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005023:Ethylene Chlorohydrin / Q000031:analogs & derivatives / Q000737:chemistry; D006735:Horseradish Peroxidase / Q000737:chemistry; D006801:Humans; D007753:Laboratories; D008967:Molecular Biology / Q000193:education* / Q000379:methods; D013334:Students; D014495:Universities
chemical_list: D005023:Ethylene Chlorohydrin; C005849:2,2,2-trichloroethanol; D006735:Horseradish Peroxidase
keywords: 2,2,2-trichloroethanol (TCE); SDS-PAGE; laboratory exercises; western blot