title: A modified gelatin zymography technique incorporating total protein normalization.
abstract: Gelatinase zymography is a commonly used laboratory procedure; however, variability in sample loading and concentration reduce the accuracy of quantitative results obtained from this technique. To facilitate normalization of gelatinase activity by loaded protein amount, we developed a protocol using the trihalocompound 2,2,2-trichloroethanol to allow for gelatin zymography and total protein labeling within the same gel. We showed that detected protein levels increased linearly with loading, and describe a loading concentration range over which normalized gelatinase activity was constant. We conclude that in-gel total protein detection is feasible in gelatin zymography and greatly improves comparison of gelatinase activity between samples.
authors.affiliation: Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, United States.
authors.forename: Julia
authors.initials: J
authors.lastname: Raykin
authors.affiliation: Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, United States.
authors.forename: Eric
authors.initials: E
authors.lastname: Snider
authors.affiliation: Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, United States.
authors.forename: Sruti
authors.initials: S
authors.lastname: Bheri
authors.affiliation: Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, United States; Department of Mechanical and Manufacturing Engineering, School of Engineering, Trinity College Dublin, Dublin, Ireland.
authors.forename: John
authors.initials: J
authors.lastname: Mulvihill
authors.affiliation: Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, United States; George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA, United States. Electronic address: ross.ethier@bme.gatech.edu.
authors.forename: C Ross
authors.initials: CR
authors.lastname: Ethier
mesh_terms: D004591:Electrophoresis, Polyacrylamide Gel / Q000592:standards*; D005023:Ethylene Chlorohydrin / Q000031:analogs & derivatives / Q000378:metabolism; D005354:Fibrosarcoma / Q000201:enzymology*; D005780:Gelatin / Q000378:metabolism*; D018093:Gelatinases / Q000378:metabolism*; D006801:Humans; D020782:Matrix Metalloproteinases / Q000032:analysis* / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D005023:Ethylene Chlorohydrin; D005780:Gelatin; C005849:2,2,2-trichloroethanol; D018093:Gelatinases; D020782:Matrix Metalloproteinases
keywords: Gelatinase; Loading controls; Matrix metalloproteinases; Trihalocompounds; Zymography