Western Blotting

This experiment will be done as the first step in determining an unknown protein. The second part of the protein determination will be done using the Western blot technique. We will use the Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) method to separate proteins by their molecular weight. This method is often used to determine the purity and weight of a protein. Electrophoresis is a method of separating proteins based on their chemical and physical properties. Gel electrophoresis has a gel support mediums that includes polyacrylamide, starch and agarose. The gel is chemically inert, it is easy to handle, and can me made to fit a desirable porosity. This makes it good to use for molecules with high molecular weights. After separating the gel, it will be stained with Coomassie Blue to visualize the protein.

In this experiment, the primary antibodies against DAT were generated from the species Rattus norvegicus (the lab rat). The secondary antibodies (Chicken Anti-RatDAT) synthesized against Rat Anti-DAT, were generated from Gallus gallus domesticus (chickens). The Chicken Anti-RatDAT antibodies were synthesized by injecting certain purified rat antibodies into a chicken. As a result, a generation of the monoclonal antibodies Rat Anti-DAT, was essential to detect the DAT segment of the protein. Likewise, the polyclonal antibody Chicken Anti-RatDAT, linkage to the Fc portion of Rat Anti-DAT. Secondary antibodies are known to assist in the sorting, detection, and/or purification of target antigens by attaching to a primary antibody, that directly attaches to the target antigen.

The filter flask, vacuum, and SNAP i.d. 2.0 were securely connected with tubing. The proteins were transferred to a membrane and was incubated with primary antibody. The primary antibody was then washed three times. Afterwards a secondary antibody was added and the same procedure was repeated. 10mL of 5-bromo-4-chloro-3-indoyl phosphate/ nitro blue tetrazolium chloride chromogenic substrate solution was added to the blot which was then wrapped with aluminum foil and labeled.

Western blotting - In Western blotting first, the macromolecules have to be separated via gel electrophoresis. The molecules now separated by electrophoresis are blotted onto either a nitrocellulose or a polyvinylidene difluoride (PVDF) membrane (a second matrix). To inhibit the binding of nonspecific antibodies to the membrane surface it is subsequently blocked. Then a complex is formed (a probe) from the protein that was transferred and an enzyme linked with an antibody. The enzyme is supplied a substrate then the 2 together should create a product e.g. chromogenic precipitate that can be detected. Detection methods with most sensitivity use chemiluminescent substrate because light is a by-product of the reaction between the substrate and the enzyme. The output of the light can be measured using a CCD camera or on the other hand, antibodies that have been tagged with fluorescents that are detected with a fluorescence imaging system can be used (Thermo-Fisher Scientific 2015).

1964). There was a statistically significant effect of time spent in the blocking solution and the fixative and staining quality. In future experiments, the time samples are placed in blocking solution and fixative should be monitored more closely and left for a sufficient amount of time to get better and more accurate results, concerning staining quality. On the contrary, there was a positive correlation between the amount of time the samples were left in the antibody solutions and staining results. The longer the samples were left in the antibody solution, the better the staining results were as attested by the statistical

After finding the concentrations of our samples using gel electrophoresis, a western blot was performed to confirm that the HCR/A had a 3xHA tag and that the HCR/T did not. I then performed an ELIZA using HCR/A and HCR/T. I bound those domains to a 96- well high binding plate. I instilled the rabbit anti-HCR/A antibody serum at different titers into each well. Next we add a secondary goat anti-rabbit HRP conjugated antibody which would attach to the rabbit antibody and would react to TMB creating a visible color. That color would be measured in a Victor spectrometer and would give us quantitative results to represent the binding of the antibodies to the HCR domains. I then purified our antibody serum using a 6% beaded agarose resin column. The plate assay was then repeated but with my purified rabbit anti-HCR/A

Traditional reagent strip testing for protein uses the principle of the protein error of indicators to produce a visible colorimetric reaction. The protein (primarily albumin) accepts hydrogen ions from the indicator causing change in color. (Susan King Strasinger, 2008)

Observe how protein biomarkers are assayed by ELISA assays beginning with serum samples, and learn how to perform these assays. (Mice sera)

The SDS-PAGE was then transferred to a nitrocellulose membrane for lab 10. This was done by creating a “gel sandwich” and passing a current through this sandwich. This membrane was then blocked with milk due to the fact that there was a large area of protein binding sites that were exposed. The milk allowed proteins to fill these areas thus preventing the antibody from binding nonspecifically. Throughout lab 10 it is also important to note the use of TBST. This molecule contained a detergent (Tween 20), and by washing our nitrocellulose membrane with it periodically we successfully reduced background staining. In order for band containing AdhP to even appear though, a V5-eptitope was placed into the protein. This epitope allowed for the Anti-V5 antibody to bind through the recognition of a 14 amino acid sequence. A secondary antibody is not needed for this blotting style due to the fact that our antibody is already conjugated to an enzyme known as horseradish peroxidase (HRP). Specific activity was then calculated via absorbance of NADH at 340nm. NADH is one of the products of the AdhP enzyme catalyzing

The purpose of the GFP tag on the gene was to detect the proteins produced by the gene through an anti-GFP antibody. Yeast strains were grown in liquid cultures at either 30°C or 42°C. Proteins were extracted from the yeast strains and amount of total protein in each extract was determined through colorimetric protein assay. An SDS-polyacrylamide gel electrophoresis was conducted. Separated proteins from the gel were transferred to a positively charged nitrocellulose membrane and the blot. Blot was then stained with Ponceau stain to detect total protein. Ponceau stain showed that all lanes contained yeast extract and showed that proteins stayed intact through the experiment. Lastly, primary antibody was added to detect proteins that were bound to the GFP tag in the western blot. After blots were exposed to light, results were obtained on a

The primary antibody is the antibody that will bind to the protein of interest. This is generated by exposing the halorhodopsin protein to a rat so that an immune response of antibodies can be generated and these antibodies are harvested from the rat. This antibody will bind to the protein of interest during incubation. The detection, however, is done with the use of a secondary antibody generated from another animal. When unbound primary antibodies are removed from the blotting sheet, the secondary antibody can be used to detect bound primary antibodies. The secondary antibody will be modified with a fluoresce tag that lights up when the secondary antibody binds with the bonded primary antibody. If there is a detectable fluoresce signal, then there is the halorhodopsin protein present in the blot. The amount of fluoresce indicates how much of the halorhodopsin gene is being generated. With the confirmation that HaloX gene was properly inserted into the genome, pain testing with optogenetics can

Two separate polyacrylamide gels were run at 100V for 60 mins. The gels were transferred to a nitrocellulose membrane and treated with primary and secondary antibodies (Methods2.6.). This was followed with three 5 minute washes with PBS-T, and scanned. The results can be seen in figure 7 as

A scientist need to demonstrate mammalian cell transfection under very sterile technique since they grow slowly and affected by contamination resulting in lower expression. In addition, the total number of mammalian cell should maintain steady and once there is confluent, cells have no more room to grow, mammalian cell culture should split using digestive enzyme and placed into new fresh media. To do so, hemocytometer used to count mammalian cell and demonstrate the cell concentration. Table 11 shows the calculation of mammalian cell concentration and for transfecting 1000,000 cell we need 1.17 ml. Following mammalian transfection, immunoprecipitation processed for FLAG-PTEN gained from mammalian cells. Anti-FLAG antibody used as ligand just as affinity chromatography to purify FLAG-PTEN. Latter on, western blotting carried out by transferring the protein from the completed SDS-PAGE to PVDF membrane as the order in figure 5. After that, incubation with primary and secondary antibody to visualize PTEN through the chemiliuminescence reaction as shown in figure 7. The figure present FLAG-PTEN in three samples the FLAG-PTEN IP, supernatant FLAG-PTEN after IP, and lysate FLAG-PTEN before IP. Lysate FLAG-PTEN before IP and FLAG-PTEN IP spouse to contain PTEN and that confirm the success of mammalian transfection. However, the FLAG-PTEN IP showed a smeared band. That may due to high protein concentration load

The sections were incubated with biotinylated secondary antibodies followed by labeled horseradish peroxidase. 3, 3’-diaminobenzidine (DAB) was used as chromogen that resulted in a brown-colored precipitate at the antigen site. Sections were counterstained with haematoxylin. Negative control sections were incubated with PBS instead of the primary antibody. Stained slides were analyzed by light microscopy (Ramos-Vara et al., 2008).

In 1959 Singer used an electron-dense protein in order to achieve ultrastructural localisation. The protein ferritin was used to tag an antibody because fluorescein lacks electron opacity. Electron microscopy could be used in immunohistochemistry as a result of this as the presence of iron in the protein makes it electron-dense (1). In 1965 Sternberger used uranium to develop the first electron-opaque heavy metal technique for ultrastructural localisation (3). Owing to the large molecular size of ferritin, ferritin-labeled antibody penetrates tissues poorly also heavy metal labeled antibody has provided insufficient increase in contrast at the sites of antigen-antibody reactions to be useful. Subsequently, the introduction of enzymes as