Electrophoretic separation of macromolecules depends upon the ability of charged molecules to move in an electric field applied in an aqueous environment (opposite charges attract!). Usually the macromolecules (proteins or nucleic acids) are placed on a hydrated solid support such as filter paper or a "gel" made of polyacrylamide or agarose. After the application of the electric field, the charged molecules migrate toward either the anode or cathode at a rate determined by their charge density, shape and molecular weight.
Proteins and Western Blotting
The membrane is covered by two antibodies, which are labeled primary and secondary. The upper antibody is coupled to the enzyme Horseradish Peroxidase (HRP). When the substrate (4CN) is added, HRP catalyzes its reaction to form a blue-purple precipitate. This enzyme-coupled color change will reveal the myosin on the nitrocellulose. Western blotting is a powerful tool used in scientific research and clinical diagnostic laboratories to identify specific proteins within a biological sample. HIV/AIDS is one of the diseases the method is used to test for.
Materials and Methods
In this experiment, the spectrophotometer absorbance is set to 562 nm. Dilute the samples and Label three small centrifuge tubes, one u, one c, and the other k for diluting the samples you extracted last week. Pipette 10 mL of each sample into a labeled small tube. Add 90 mL of distilled water to each. From here, Pipette 100 mL ml of each standard and unknown into the appropriately labeled cuvette. Then, 2.0 ml is added from the reagent being used before covering with Parafilm, blend properly and Cover and incubating tubes for 30 minutes at 37o C in water bath. Cool tubes to room temperature. Cool tubes to room temperature. From this point, the results of the experiment are obtained.
Starting with samples from the last week, an extract from an unknown species (u), prepared, followed by A known sample of real crab (c), and finally a known sample of fake crab (k). From this point, the gel is opened and set up in the electrophoresis chamber as shown by the instructor. Using the gel loading tips, the amount calculated above is loaded into the wells. To remove the gel from the cassette, the cassette is laid on top of the bench top, with short plate up. Cautiously, the gel plates are pried apart with the special opener. The gel must not be touched with ungloved hands.
The first procedure in this case was the one used to detect myosin. To start, Orient the nitrocellulose membrane using the pre-stained standards as a guide. The Wet membrane is soaked with Wash Buffer. Once this is done, the membrane is placed with the pre-stained standards looking up in blocking solution after which it is placed on a rocker for 15 min. After this add 10 ml anti-myosin antibody (in blocking solution) and place on the rocker for 20 min. at room temperature. The color development is monitored and then the developed blot photographed.
The second procedure in this experiment is to calculate the molecular weight of myosin. The prestained protein standards are used in this case to determine the molecular weight of the proteins that are found in the SDS-PAGE gel. The distances of the 10 bands in the protein standard is measured and then recorded. Once this is done, the distance that the myosin has moved is measured and recorded. The log MW is plotted on the y-axis of the graph, while the x-axis is plotted with the distance. From the graph, the molecular weight of the myosin can then be determined.
After the concentrations of the samples were measured, the absorbance for the real crab was 0.285. Additionally, the absorbance for the fake crab specimen (K), was 0.306, while the absorbance for the unknown specimen was 0.440. The results for the concentration of BSA standards versus the absorbance are shown in the picture found in the tables and figures section. Additionally, the plot of the graph obtained by the pair of result s is found in the same section and labeled excel for Lab 1.
The results for the second lab are shown I the picture shown in tables and figures sections and it is labeled lab 2. In the picture, it is clear that the smaller proteins tend to migrate faster when compared to the larger proteins. They are shown to have longer strips than the larger proteins. Additionally, the bands for the fresh crab meat are not easily distinguished in the picture.
From the ladder obtained from the picture of the protein standard equipment, it is seen that the sample number two represented fresh crab meat. The sample number three, on the other hand, represents the imitation crab. The results obtained for the purpose of drawing a grapgh of log MW on the y-axis and the distance migrated on the x-axis is shown in the picture of the lab notebook in the tables and figures section. From the results, the myosin band migrated 28 millimeters. This was equivalent to 1.45 in the log scale.
After the results obtained in the section above were analyzed, the graph of best fit was drawn. It was seen that the graph was a straight line. Hence, due to the fact that the graph was a straight line, it can e used to determine the protein concentration of the fake crab k and that of the unknown sample. It can be done by using the protein concentration of the samples, then measuring the BSA value for each sample. After this measurement is obtained, result is compared to the standard value. Quantification of proteins is extremely important nowadays. It is because metabolic engineering requires an accurate quantification when coming up with models. Hence, the method used in this lab experiment is not the most accurate method. However, it is a reliable method when it comes to performing the analysis of protein content in foods.
In this lab, one of the key observations was the fact that the smaller proteins move faster than the larger proteins. These smaller proteins seem to navigate the mesh at a much faster speed than the bigger proteins, and hence they can be separated by their speeds on the mesh.
From the third lab results, it is clear that the unknown sample represented fake crab. This is because the fake crab has myosin. The two methods used produced results that support the same conclusion. Hence, it is clear that western blotting is a much easier method than the Coomassie staining. It is highly applicable in many situations. However, the implementation of the method is vital to its success. One has to be careful when loading the gel and the suitable buffer for homogenization should also be considered.
Figures and Tables
The graph below shows the absorbance value against the protein concentration.
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