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The Use Of The Western Blot Analysis To Identify Four Unknown Species Of Fish

1373 words - 6 pages

Protein extractions from unidentified fish samples were separated according to the molecular weights by SDS polyacrylamide gel electrophoresis. Since some of these proteins are shared between fishes, phylogenetic evaluation was reached. Western blot analysis was used to identify four unknown species of aquatic animals via comparison of actin/myosin bands. According to the results of this assay, the best estimate is that the unidentified aquatic animals are specimens of salmon, tilapia, cod, and shrimp, respectively.
Introduction
Western blot has been a revolutionary technique for identifying the expression of proteins within relative molecular biological samples that shared the same ancestor. Moreover, the sensitivity and specificity of the western blot (Immunoblotting) enables it a common technique for determining specific protein levels in clinical samples. Since the antibody specific to the antigen immunospecificity), it enables the target protein to be identified. Western blotting can produce quantitative data about that protein, which in this case the difference between bands in each of the protein samples. The western blot is an analytical technique used to detect specific proteins in the given sample of tissue homogenate or extract. The proteins are then transferred to a membrane (in this case, nitrocellulose), where they are stained with antibodies specific to the target protein [1] [2].
This experiment designed to analyze the phylogenetic of four unknown different fish muscles samples in order to predict the type of the aquatic creature. In this study, actin and myosin proteins were analyzed on account of their fairly high and constant expression in the animal muscles. Since the actin and myosin proteins are the two major components in muscles movement, therefore, the amount of actin and myosin in the animal helped in restricting the phylum and predicting the type of the animal. Conducting the comparative expression analyses were assisted by using the reference proteins control (Kaleidoscope protein standard) and Actin/Myosin positive control.
Materials and Methods
Protein Isolation
Four pieces of fish muscles were cut and transferred to four labeled microcentrifuge tubes, and mixed with 250µl of Laemmli buffer to lyse the cells. Tubes were vortexed. They were incubated at room temperature for 5 minutes. Then tubes were centrifuged to pellet out the particulate substance. The supernatant was transferred to new tubes.
Bradford protein assay
Appropriate dilution for each fish sample was determined, by preparing 1:20 dilution of fish protein to PBS buffer for each of them. The protein concentrations for BSA are shown in table 1. Each cuvette contained 1 ml of Bradford reagent stock solution, 20µl of the appropriate BSA standard, and 20µl of the of the unknown fish protein. Samples were incubated for 5 minute. After incubation, absorbance was measured for each sample at 595 nm. Standards were used to create a standard curve so that unknown...

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