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
This technique separates Rubisco samples based on their size. The electrophoresis has a positive and a negative end. Positive charge proteins are loaded from the positive end and migrate towards the negative end. Negative charge proteins are loaded from the negative end and migrate towards the positive end (Sakthivel & Palani, 2016). The sample that contained the highest molecular weight of Rubisco will travel the shortest distance on the gel while the protein with the smallest molecular weight will travel the longest distance (Sakthivel & Palani, 2016). The size proportion of each Rubisco molecule correlates with the distance traveled. Rubisco will be in its purest form after running through SDS-page since each technique will increase the purity of the protein. If the salting out, the ion exchange and the SDS-page protein isolation techniques are performed on protein Rubisco, then it is purified and separated by solubility, charge, and size. The rationale of this experiment is to isolate the purest form of Rubisco so that it can perform carbon fixation at an optimal
Gel electrophoresis method. Qualitative analysis shows protein concentrations in kidney, heart, and liver. 1-6 are kidney tissue. 7-14 are liver and 15-19 are the heart tissue.
In Activity B, Running Agarose Gel Electrophoresis, began by preparing and
After the substrate solution was added, five drops of the enzyme were quickly placed in tubes 3, 4 and 5. There were no drops of enzyme added in tubes 1 and 2 and in tube 6 ten drops were added. Once the enzyme solution has been added the tubes were then left to incubate for ten minutes and after five drops of DNSA solution were added to tubes 1 to 6. The tubes were then placed in a hot block at 80-90oC for five minutes. They were then taken out after the five minute period and using a 5 ml pipette, 5 ml of distilled water were added to the 6 tubes and mixed by inversion. Once everything was complete the 6 tubes were then taken to the Milton Roy Company Spectronic 21 and the absorbance of each tube was tested.
Colorimetric assay is a process of determining a concentration of a solution based on absorbance of light. The purpose of this lab is to determine if the Bradford assay is an accurate way to determine an unknown concentration of two samples of protein. The Bradford assay is done by measuring wavelength of light passing through a cuvette filled with Bradford dye and concentrations of PBS and proteins. After the cuvettes are mixed they are placed into a spectrophotometer to measure wavelength. The wavelength given will be used to plot a standard curve based on concentration (x-axis) and wavelength (y-axis). The standard curve is then used to measure an educated guess on the concentrations of unknown protein concentrations. We hypothesized that if we use the Bradford assay and colorimetric spectrophotometry we can determine an accurate concentration of two unknown concentrations of proteins. The results of this lab failed to reject our hypothesis based on accurate measurements of protein concentrations. The standard curves are drawn with a linear increasing slope. The Bradford assay is an accurate way to demine the concentration of an unknown concentration.
Observe how protein biomarkers are assayed by ELISA assays beginning with serum samples, and learn how to perform these assays. (Mice sera)
This Lab Report is an analysis of the results of a two-part experiment. In the first part, we used a gel filtration column to separate the components of a mixture composed of protein and non-protein molecules. By doing so we hoped to obtain fractions that contained single components of the mixture, while also gaining insight into the relative molecular weight of each component compared to each other. We would then plot these fractions onto nitrocellulose paper in order to determine which fractions had protein. In the second part, we would use the fractions which we had determined had protein to conduct an SDS-PAGE. By doing so we hoped to determine an estimate on the molecular weight of the proteins present in each fraction by comparing it to a tracker dye composed of a variety of molecules of differing molecular weight.
ELISA works on the principle of an antigen binding to specific antibody (lock and key), which can be used as a way to identify quantities of proteins in a small sample of fluid. The specific proteins used in an ELISA are estimated quantitatively. The ELISA test is carried out by incubating the serum that contains the antigen of interest with antibody’s within a well, in order for the antibody’s to bind with the specific antigens. The plate is then washed with a mild detergent in order to remove any proteins that have not been bound. The washing of the plates is carried out between every step in order
For the second part of the experiment, one had to use the knowledge learn from viewing protein molecules in FirstGlance in Jmol to analyze the protein PDB ID: 4EEY. The analysis of this protein was done using the RSCB protein data bank (PDB) at (http://www.rcsb.org/pdb/home/home.do).2
The last part of this procedure, antibodies will be used to detect one specific protein from others on the membrane. Incubate the membrane with 10 ml of primary antibody for 10-20 minutes and then place on a rocking platform. Rinse the membrane quickly after with wash buffer on a rocking platform. After three minutes is up, discard the wash and incubate the membrane with 10 ml of secondary antibody for 5-15 minutes on the platform. Rinse the membrane again shortly after and wash the membrane for three minutes. Next, discard the wash and add 10 mL of HRP color detection reagent. Incubation will occur after this for 10-30 minutes. Once it is done, rinse the membrane twice with distilled water and blot dry.
After the treatment with WECU, the cells were harvested, lysed, and the protein concentrations were quantified using a Bio-Rad protein assay as described in a previous study [You et al., 2017]. In a parallel experiment, the mitochondrial and cytosolic fractions were isolated using a mitochondria isolation kit according to the manufacturer’s instructions. For Western blotting, equal amounts of protein samples were electrophoretically transferred onto polyvinylidene difluoride membranes (Schleicher & Schuell, Keene, NH, USA) following electrophoretic separation on sodium-dodecyl sulfate (SDS) gel. After blocking with TBS-T buffer [20 mM Tris (pH 7.4), 150 mM NaCl, 0.1% Tween 20] containing 5% skim milk, the membranes were probed with specific primary antibodies at 4 °C overnight, and then incubated with the appropriate HRP-conjugated secondary antibodies for 2 h at room temperature. The protein bands were detected using an ECL kit as per the manufacturer’s instructions.
The labels associated with the resulting proteins from previous steps were rinsed and removed. USP7 was purified and utilized in sedimentation trials in varying concentrations. Absorbance analysis was carried out after the samples were centrifuged. EBNA1 was purified, ubiquitinated, and combined with USP7, which was purified in previous steps. SDS-PAGE was used to disrupt the protein interaction and separate them. The separated fragments were then used in a western blot, and analyzed after probing. The affinity of USP7 for EBNA1 and p53 was tested using a stepwise combination of titration, incubation, and fluorescence analysis. Lastly, amino acid residues of EBNA1and USP7 were studied by gel filtration and size-exclusion chromatography.
The major use of Enzyme Linked Immunosorbent Assay (ELISA) is to titrate antibodies against specific antigens (Crowther, 1995). This method involves an enzyme, which reacts with a colourless substrate to form a coloured product, is covalently linked to a specific antibody that recognises a specific antigen. If the antigen is present, the antibody-enzyme complex will bind to it, and the enzyme component of the antibody-enzyme complex will catalyse the reaction which results in a coloured product (Berg et al, 2002). The presence of the coloured product indicates that an antigen is present. The ELISA, which is rapid and convenient, can detect less than a nanogram (10-9 g) of a protein and can be performed with either polyclonal antibodies, which are antibodies that can recognise multiple epitopes on any one antigen or monoclonal antibodies, which are antibodies that can only detect one epitope on the antigen (Guo et al, 2006). However, the use of monoclonal antibodies gives more reliable and accurate results due to their specificity as monoclonal antibodies are very effective at acting as the primary antibody in an assay, or for detecting antigens within a tissue. Also, they will usually result in reduced background staining compared to that of polyclonal antibodies.
ELISA or Enzyme-linked immunosorbent assay is a biochemical technique used to check for the presence of a target protein of interest in an experimental sample and how much of protein is present (Voller et al, 1978). ELISA is considered an
Sitewise analysis was carried out and the mean and standard deviation of various RNA and protein values at different sites are shown in figure 6.4. The results showed that there was no significant difference between sites with respect to the mean RNA and protein values.