Western Blottography Lab Report

To perform this test, a small drop of water is placed on a clean microscope slide. A metal loop that has been properly sterilized in the blue flame and allowed time to cool is used to

The purpose of this experiment is to perform a procedure known as SDS-PAGE (sodium dodecyl sulfate - polyacrylamide gel electrophoresis) in order to determine protein profiles for seven fish species: Yellow Tuna (Thunnus albacares), Thresher Shark (Alopias vulpinus), Paiche (Arapaima gigas), Swordfish (Xiphias gladius), Catfish (Ameiurus catus), Coho Salmon (Oncorhynchus kisutch), and Rainbow Trout (Oncorhynchus mykiss). The gel electrophoresis separates the fish muscle proteins according to their molecular weights, which allows one to determine a profile for each specie.

Blood is a highly specialized complex connective tissue with a unique fluid of variable composition circulating through the heart, arteries, capillaries, and veins, known as the vascular system of the body (???). Blood approximately accounts of 8% of the body weight, with female body consists of 4-5 litres while males have around 5-6 litters. This variation is mainly due to the difference in body size between men and women (@@@). Human blood has a pH of 7.35-7.45, this makes the blood to be basic in nature. The viscous nature of blood makes it more resistant to flow than water. Unless blood is viscous, it can strain the heart and lead to severe cardiovascular problems (@@@). Due to the presence of high oxygen in arteries,

Next, gather 0.5 ml of the protein extract and save this portion in a separate container. This will be used for the preparation of the membrane.

After 30 seconds we expelled the solution back into the cup and swirled gently to mix cells. Second, we transferred 1000µL of the solution into our labeled microcentrifuge tube. Third, centrifuge for 90 seconds at full speed then pour off most of the supernatant into the paper cup without disturbing the pellet. Fourth, we have to resuspend the cells then withdraw 30µL of the cell suspension and add it to the PCR tube with 100µL Chelex. After we put the labeled tubes in the thermal cycler which was set to 99ºC for 10 minutes, once done shake vigorously. Fifth, we centrifuged the sample for 90 seconds at full speed once done we transferred 30µL of the clear liquid into a new 1.5mL tube and labeled it with our assigned number. Store on ice until ready for it in part

Divine Woodfin Ms. A. Allison Honors Chemistry September 18,2015 What is the effect of different impacts on blood stains? The purpose of this project is to determine the effect of different impacts of blood on the stains that they leave. I chose to conduct this experiment as an extension of a previous experiment which only dealt with the amount of blood splatter within a given height.

Paper towels are invaluable product for cleaning up everyday spills in every household. The United States is the largest consumer of paper towels. In the United States, 50% more paper towels are consumed than Europe and five times more than Latin America. More importantly, we use it daily in my household. With this in mind, I wanted to determine which product on the market absorbs the most and cost effective.

The Bradford and Lowry assay are two color changing proteins with similar processes. Color changes of the solution can be measured using a device called a spectrometer. In lab, the two assays can be used to determine the amount of protein in a solution. This can be done in an experiment by reacting a specific reagent with amino acids or peptide bonds present in the proteins. This produces a color change in the solution depending on the amount of peptide or amino acid reactions with the reagents.

In this method, cells are incubated with antibodies conjugated with fluorescent dyes such as FITC (fluoroscein isothiocyanate), which emits green wavelengths of light; PE (phycoerythrin), or Texas Red, both of which emit red wavelengths of light. Newer fluorescent dyes emit at yellow and blue wavelengths as

The purpose is to transfer biological samples from a gel to a membrane and their subsequent detection on the surface of the membrane. An antibody is used to specifically detect its antigen for protein analysis. The specificity of the antibody-antigen interaction enables a target protein to be identified in the middle of a complex protein mixture.

Use a pipette and collect 23 Paramecium cells from their culture. Squeeze the Paramecium cells from the pipette into the electrophoresis chamber. Let the Paramecium adapt to the new environment for 1 minute.

If the colour changes to mauve/purple then this shows protein is present. Lipids Method: 1. Add 1cm3 of ethanol and then same with oil into a test tube.

No water or SLB will be used, instead samples will now mix with Beta-Mercaptoethanol (2-ME) 6ul for G0 and G3, and 3ul for W3,W4, E2,and E3, and 15ul of each (GCE, G3 and ladder only load 5ul) will be loaded into the gel. Run the electrophoresis at same voltage and time as last time. Once the dye front is at the green gasket, turn off the machine, and pull the plates apart the same way take off the stacker, and notch the corner next to the ladder. Set up the western blot machine, three wet filter paper, on the positively charged base, on top of that, a wet nitrocellulose paper then the gel, make sure there are no bubbles, and three more wet filter papers on top of that, lastly the lid, which has the negatively charge base on it, lock the lid, and allow to run over night. After the process is done, use forceps to take apart the layers of the blot, to reveal the nitrocellulose paper. Place the paper in a Tupperware, adding 20 ml of Ponceau S stain, shake (rocking motion) for about 1-2 minutes discard the stain, and rinse the paper with water, multiple times until red bands appear. Mark the ladder with pencil. Next place the paper in a blue Tupperware, with a clear lid, and add 30 ml of 5% nonfat dry milk/ TBS solution, and place on the shaker for thirty minutes. When the time is up discard the Blocker (dry milk) and add 7ml of mouse IgG anti-Xpress epitope Mab Solution, and place back on the shaker for 45 minutes. Once the 45 minutes are up, pour out the primary antibody, and add 30ml of 0.05% Tween 20/TBS and place back on the shaker for five minutes, after, pour out the wash and repeat two more times. Next, add 7ml of Sheep IgG anti-mouse IgG conjugated horse radish peroxidase polyclonal anti-serum solution and place on the shaker for 45 minutes. Once 45 minutes have passed, pour out the solution, and repeat the wash step from above, again three wash step. After the third

On our SDS-PAGE, our protein standards were separated alongside our protein samples which allowed determination of the sizes of our protein in our sample. SDS denature proteins and coats them with a negative charge allowing for their electrophoretic separation by polyacrylamide gel electrophoresis (PAGE). As you can see, after being stained with the coomassie blue, we could detect proteins of various sizes and our unknown protein expressing GST was detected in our Pure B sample (Figure 2). The distance travelled from the wells on the SDS-PAGES by our protein standards were measured and compared to our protein ladder in Daltons (Table 2), from which we could construct our semi log plot. On the semi-log plot, our unknown protein within our Pure B travelled 27mm from the well with a size of 29,000 Dalton (Figure 3). To identify the specific protein in the gel, the western blotting method was used to detect which of the cultures (A or B) contains GST (Figure 4). After the cassette was taken out of the electrophoresis unit, colors of our protein standards appeared on top of our membrane surface. After blocking, primary antibody incubation, and secondary antibody incubation, we could clearly identify our GST in our Pure B and Crude B (Figure

The next step involves incubating the culture medium for 48 hours at 37° Celsius. If the chemical being tested is