title: Viability of a FRET dual binding technique to detect calpastatin.
abstract: We have been investigating a fluorescence dual binding biosensor to detect calpastatin. Calpastatin is a protein found in meat and it is a regulator of meat tenderness. The ability to accurately predict the calpastatin concentration of beef with a biological sensor at the time of grading would lead to a more accurate assessment of the overall palatability of beef when it reaches the consumer. Meat can then be labeled as tender or tough, which would greatly enhance meat processors' ability to grade meat, allowing them to recover lost revenue. The biosensor technique utilized the chemical transduction principle of fluorescence resonance energy transfer (FRET). FRET requires the use of two fluorophores, termed a donor and acceptor. In this study, the donor fluorophore was conjugated to the protein, mu-calpain, while the acceptor fluorophore was conjugated to a monoclonal antibody. The results showed that in the presence of calpastatin, the labeled mu-calpain and antibody would bind to calpastatin, reducing the distance between the two proteins and eliciting a measurable change in fluorescence. The FRET dual binding technique was tested in heated and unheated meat extract, and a limit of detection for calpastatin was 120 ng/ml in diluted heated meat extract with no significant response in the unheated meat extract. Stable response times were achieved within 5 min. The proof-of-principle of utilizing a FRET dual binding technique to detect calpastatin in heated meat extract has been established.
authors.affiliation: Department of Biological Engineering, 250 Ag. Engineering Building, University of Missouri, Columbia, MO 65211, USA. grantsa@missouri.edu
authors.forename: Sheila A
authors.initials: SA
authors.lastname: Grant
authors.forename: Richard C
authors.initials: RC
authors.lastname: Stringer
authors.forename: Seth
authors.initials: S
authors.lastname: Studer
authors.forename: Darcy
authors.initials: D
authors.lastname: Lichlyter
authors.forename: Carol L
authors.initials: CL
authors.lastname: Lorenzen
mesh_terms: D002135:Calcium-Binding Proteins / Q000032:analysis*; D005240:Feasibility Studies; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes; D005504:Food Analysis / Q000379:methods*; D008460:Meat / Q000032:analysis*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D002135:Calcium-Binding Proteins; D005456:Fluorescent Dyes; C033668:calpastatin