What Additional Confirmatory Tests Should Be Done?

b. Attached to the secondary antibody is an enzyme such as peroxidase or alkaline phosphatase. These enzymes can metabolize colorless substrates (sometimes called chromagens) into colored products. After an incubation

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.

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.

Know the HIV status of your partner(s). If your partner is HIV-positive, make sure he or she has an undetectable viral load on HIV medicines.

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

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.

On a thin chromatography plate, five spots were placed ( as shown in table 2) and the plate was developed using chloroform/methanol. This was later visualized with dragendorff’s reagent under the UV light. All separated components were observed, identified and recorded.

Furthermore, the PCR product will then be purified, in order to digest and ligate into a vector and transform E.Coli cells, in later parts of the experiment, to induce bioluminescence in those E.Coli cells [1].

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 following procedure dealt with a chromatogram. The materials needed are: a pencil, safety goggles, scissors, chromatography paper strip, capillary tube, spinach plant pigment extract, test tube, cork stopper, graduated cylinder, chromatography solvent (alternative isopropyl alcohol), metric ruler, stopwatch or clock with a secondhand, hook/fashioned paperclip, paper towels, test tube rack, and mortar and pestle. First we obtained a strip of chromatography paper and cut it so it would fit inside a test tube (with it barely touching the bottom of the tube). Also, when touching the strip, touch the sides only. Then we attached (firmly) the top of the strip to a hook (or fashioned paperclip at bottom of the cork stopper). Make sure it fits in the test tube. Next we used the pencil to draw a faint line across the strip two centimeters from the bottom tip of the strip. We placed the cork and strip in place, and we put a mark on the test tube one centimeter below the top of the stopper.

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.

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).

There are several different HIV tests that are administered and an even a greater number of testing sites. The most common HIV test is giving is antibody screening. The screening test the reaction of your own antibody makes against HIV. The procedure can be done by two forms; which are blood or orally fluids. Since time has evolved so outstandingly; you don’t necessarily have to leave the comfort of your own home anymore. There are home

It is an automatic whole slide scanner that is comprised of the following components: an optical focusing system, a CCD camera, a Z-focusing system, an XY-translational stage, bright field illumination, a multi-color fluorescence excitation and emission filter system, a microprocessor control board, and a computer. The user can upload up to 8 slides for processing and can view images in up to 5 fluorescent colors. Vanguard scans the entire surface of the microfluidic slide with a series of fluorescence filters that are defined for a given assay and acquires images. A standard assay includes capture of images using filters for DAPI (nucleus), Alexafluor 488 (pan cytokeratin [PanCK], an epithelial marker) and Alexafluor 594 (CD45, a leukocyte marker). Additionally the user can capture data with filters for Cy3, Cy5 or PE and APC. After data acquisition is completed, the images are analyzed for any event where DAPI and cytokeratin are within a specified space in the slide, i.e., indicating the possible presence of a cell with a nucleus that expresses cytokeratin. Images from each fluorescent color, as well as a composite image are presented to the user in a gallery for CTC calls. A cell is classified as a CTC when it is DAPI+, PanCK+ and CD45-. A check mark is placed by the operator next to the composite images and the software tallies all

These tests detect the antibodies for each patient to these HIV viruses inside the blood. These are two tests. Both tests have to be positive to confirm the HIV infection to be totally true. To get better results, patients can take the complete blood count (CBC) and/or the white blood cell differential. Both tests can show all the abnormalities inside the blood of the body. Yet, if the results fully confirm that it is positive, it is important to get treatments as soon as possible.