Gel Myth Tube Analysis

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The eleven tubes are also known as the “standards.” The twelfth tube will consist of the isolated protein sample obtained in (a). After preparing the dilutions of both standards and unknown, 5 μL of each standard solution and isolated protein sample is transferred onto individual wells from a multi-well plate. Additionally, all standards and unknown solutions are placed in replicas onto two individual rows of the multi-well plate in order to obtain consistent and accurate results. After all solutions are placed in each well and in two rows, 250 μL of Bradford-Coomassie is added to both standards solutions and unknown protein solutions. Moreover, the multi-well plate is then analyzed using a Bio-Tek plate reader where the absorption values…show more content…
After determining the concentration of the unknown protein sample (_mg/mL), the isolated protein sample will now be separated based on protein size by gel electrophoresis. Gel electrophoresis allows the movement of a molecule with a net charge using an electric field. In addition, the gel serves as a molecular sieve, which reinforces the separation of proteins. Thus, protein molecules that are large in size are usually found at the top of the gel, whereas the protein molecules that are smaller in size are found at the bottom of the gel. This is because the protein molecules that is smaller in size more easily and faster through the pores of the gel where the large ones move more slowly. Consequently, the type of gel used in this experiment is a polyacrylamide gel electrophoresis (PAGE) which is a vertical gel flowing top to bottom for the proteins to move through the positive pole, which the bottom of the gel. The isolated protein sample was then coated with sodium dodecyl sulfate (SDS), which serves as an anionic detergent and purposely coats the proteins with a negative charge. This allows the proteins to move through the gel from the top of the gel (negative pole) to the bottom of the gel (positive pole). Moreover, β-Mercaptoethanol is also added to the isolated protein sample as it serves as a reducing agent by breaking disulfide bridges allowing the proteins to separate easily throughout the gel based on size. Furthermore, upon loading the isolated protein sample to…show more content…
Thus, primary and secondary antibodies are used to recognize the antigen of interest (CRP). The secondary antibody is used to recognize the constant regions of the primary antibody. In this case, the primary antibody is the antigen. Thus, an immunoblotting analysis is done. An immunoblotting analyses allows the antibody to bind to immobilized antigen in vitro. Additionally, immunoblotting uses nitrocellulose paper for the proteins to bind to as the proteins natural stick to the nitrocellulose paper. The nitrocellulose membrane is the block in the solution containing milk protein (blotto). This blotto binds to the nitrocellulose and prevents non-specific binding of antibodies. Consequently, the primary antibody (antiserum or anti-CRP) is incubated with blotto at room temperature. After incubation, unbound antibody is washed away fore adding the secondary antibody. The secondary antibody usually alkaline phosphatase is added in with blotto and incubated at room temperature. Once again unbound antibody is washed away before adding the substrate. The substrate is then added and the signal is allowed to develop as the substrate changes in color from yellow to purple. Therefore, the primary antibody must be present for the secondary antibody to bind to. The change in color from yellow to purple only forms when the entire complex is present as the substrate precipitates. As a result, the
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