Requirements for the application of protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis and randomly amplified polymorphic DNA analyses to product speciation

Abstract

Raw, cooked, fried, smoked and gravad (brine-cured) products were analyzed by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of proteins and by randomly amplified polymorphic DNA (RAPD) in order to identify the species used in their manufacture. The discriminatory power of SDS-PAGE was dependent primarily on the composition and secondarily on the size of the gels: the Laemmli buffer system with 15% acrylamide and 0.087% piperazine diacrylamide separating gels resolved more discriminant protein bands than any of the commercial gels tested. Some of the processing conditions induced alterations in the protein patterns that made identification dubious. Differentiation even between closely related species was easier by RAPD than by SDS-PAGE. Neither the processing conditions nor the tissue from which the DNA was extracted had a significant effect on the RAPD profiles. For identifications based on SDS-PAGE, one should use an optimized gel composition and separate the sample under analysis in the same gel as the references. For RAPD-based identifications, the unknown sample should be amplified together with reference samples and separated in the same gel.

Genetic Identification of Antimonate Respiratory Reductase in Shewanella sp. ANA-3

“…Then the target protein was eluted with a HEPES buffer containing 100 mM imidazole (pH 7.4) and detected using sodium dodecyl sulfate (SDS)−polyacrylamide gel electrophoresis (PAGE). 23 Catalytic Kinetics of ArrAB. To measure the activity of ArrAB reduction for Sb(V) and As(V), a kinetics study was carried out following a previous report.…”

“…The soluble supernatant was mixed with 1 mL of pre-equilibrated Ni–nitrilotriacetic acid (Ni-NTA) His-Bind Resins (7sea Biotech, China) and gently agitated at 4 °C for 2 h. The resin was transferred into a 10 mL gravity-flow column and washed by 3 mL of HEPES buffer with 60 mM imidazole (pH 7.4) to elute the miscellaneous proteins. Then the target protein was eluted with a HEPES buffer containing 100 mM imidazole (pH 7.4) and detected using sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) …”

Methods of gadoid fish species identification in food and their economic impact in the Czech Republic: a review

“…For the fish species identification of the mixture of two or more fish species, the authors recommended the use of the speciesspecific primers. Martinez et al (2001) carried out the comparison of SDS-PAGE and RAPD for the several fish species determination and Gadus morhua was included in this study. RAPD analysis was considered to be more discriminating technique, with the efficiency to identify fish species in processed (cooked, fried, gravad and smoked) fish samples.…”

Feed Authentication as an Essential Component of Food Safety and Control

“…Traditional (and official) methods for identifying the species sources of animal products rely on the identification of species-specific proteins or groups of proteins by such techniques as isoelectric focusing (IEF), capillary electrophoresis (CE) and immunoassay (Rehbein, 1990;Martin et al, 1991;Zerifi et al, 1991;Sherikar et al, 1993;Knuutinen and Harjula, 1998;Martinez et al, 2001). However the high temperatures necessary to treat animal material during rendering (Council Directive 90/667/EEC; Commission Decision 92/ 562/EEC) mean that electrophoretic and immunological techniques cannot be used to identify species in meat-and bonemeal since their proteins are completely denatured.…”

“…Another technique is PCR amplification of anonymous DNA regions by the randomly amplified polymorphic DNA (RAPD) method; this has been shown to identify the animal species present in variously treated and cured human food products (Martinez et al, 2001;Rego et al, 2002).…”