Enzyme Linked Immunisirbent Assay

Protein Assay: The Pierce BCA Protein Assay (Thermo Scientific) is a detergent-compatible formulation based on bicinchoninic acid (BCA) for the colorimetric detection and quantitation of total protein concentration. A series of standard solution of Bovine Serum Albumin (BSA) ranging from 0-2000 µg/ml was prepared from a stock solution of 2 mg/ml BSA. 25ul of diluted crude (1:500, 1:250), desalted (1:100, 1:50), and 6 peak fractions from cibarcon blue column (1:10, 1:5) were loaded in microplate along with 175ul of BCA working reagent. Microplate was incubated for 30min at 370C and then the absorbance was measured at 562nm.

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.

Eisenthal, R. & M.J. Danson (Eds.) (2002). _Enzyme Assays: A Practical Approach_. United Kingdom: Oxford University Press

The homogenates provided were made by homogenizing tissues in a sucrose phosphate buffer in a 1:20 ratio. The protein concentration in bovine cells was measured by diluting the homogenate with a 1:5 ratio; 50 microliters of homogenate and 200 microliters of water. Then 5 known protein concentration samples which were 0.4, 0.8, 1.2, 1.6, 2.0 mg/ml of bovine serum were used to determine absorbance with a spectrophotometer. Two additional samples were made; one was blank and the other was for the specific homogenate sample. Then 3 microliters of bradford assay reagent, which indicates the amount of protein present

Andra Larade Bio 30 B7 11/20/15 Liver Enzyme Lab The Effect of pH and Temperature on the Efficiency of Catalase Abstract: In order to determine how pH and temperature affect the liver enzyme catalase, an experiment was conducted. Catalase breaks apart hydrogen peroxide producing water and oxygen.

The enzyme-linked immunosorbent assay (ELISA) is a common laboratory technique used to measure the concentration of an analyte (usually antibodies or antigens) in solution. In the practical anti-BSA antibodies that had undergone serial dilutions were added to a BSA solution in an ELISA plate with goal of seeing how the concentration of anti-BSA antibodies would affect the colour change of the BSA solution. The results clearly showed a direct correlation as the more diluted the anti-BSA antibody solutions became the lower the Wavelength readings at 405nm, which showed that there was less of a colour change.

The results are organized in sections by order to which the experiments were performed. There are a total of four sections that is represented by a major heading and is shown in bold print that corresponds to the four experimental approached used in this study. Each section also contains either a table or a figure or a combination of both that corresponds to each experiment. The first section was to first figure out the best possible combination of these monoclonal antibody cocktails (ZmAb) and MB-003 by using laboratory animals most specifically guinea pigs and NHP’s (rhesus macaques) to test which combinations of MB-003 and ZmAb are the most effective and can be used for further experiments. To test the components of MB-003 (mAbs c13C6,

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

Overall, ELISA is a good serological method in immunodiagnostics, especially for less developed communities due to its cost and efficiency. However, it does come with limitations, mainly due to the economic status of the geographical areas of infection and the infections the individuals are commonly exposed to. As well as the effect that multiple infections can have on the sensitivity and specificity of

Second, for the western blotting, the primary antibodies that used to recognize the protein of interest (FLAG-PTEN) are rabbit anti-PTEN antibodies and the secondary antibodies that bind to the primary antibodies and convalently linked substrate to produce visible signal are goat anti-rabbit conjugated HRP (horseradish peroxidase) antibodies; for immunoprecipitation, the antibodies used to immobilize protein of interest are anti-FLAG monoclonal antibodies from morine cell. As introduction above, those antibodies come from different species. In order to be successful in western blotting, the primary antibodies recognize the protein of interest as well as bind to it. Consequently, rabbit anti-PTEN antibodies were used to bind to FLAG-PTEN. Meanwhile, goat anti-rabbit conjugated HRP antibodies, the secondary antibodies,

1) Seroconversion using two serum specimens collected at an interval of at least 14 days.

To confirm the blood is human blood, I would use the ELISA test and the Kastle-Meyer test. Once the tests are complete and confirm human blood, I will analyze the splatters on the wall. Stain B looks as though someone attempted to wipe it off, as it seems to be smeared. Stain A appears to be moving left. Stain D and C look like stains from high impact. I would guess a gun was used at this scene, and that is what created the splatters on the

Described by Coligan et al (2005), Glutaraldehyde coupling was needed to conjugate the two peptides individually to bovine serum albumin. BSA dissolved in 500ul of 0.1M borate buffer pH10 was manipulated to mix with the air-oxidized peptide solution that was already prepared taking note that the mixture has to be on a 1:10peptide-BSA ratio. Moreover, until a 1:10:1 peptide-BSA-glutaraldehyde ratio was achieved, the peptide-BSA mixture was combined with 1% glutaraldehyde. Appendix B includes the computations for the preparation of antigen conjugation. After, the mixture was incubated for 2 hours at room temperature and was diluted with distilled water until a 1mg of peptide in 1ml of the peptide-BSA-glutaraldehyde

While Immunoassay testing is the most popular and cost effective way to drug test, it is not the most accurate. Cone and Menchen (1987) conducted a study involving the KDI Quik Test Drug Screen, which is a drug screen that relies on immunoassay testing alone, and has the ability to detect the use of cocaine, morphine, phencyclidine, amphetamines and other metabolites associated with illicit substances (p. 276). The study focused solely on whether the test indicated a presence of benzoylecogonine, which is the metabolite of cocaine. Overall, the results concluded that the KDI Quik Test was not an accurate test by itself and that positive or negative results would still need to be validate with further testing in a lab. Additional results from the study indicated that immunoassay testing could lead too false positives due to ill trained staff administering the test. In my professional experience, as a Certified Drug and Alcohol Counselor in the State of Delaware, this is true; there were many times when I conducted a urinalysis utilizing the immunoassay method of testing only to decipher that the results were inconclusive, which were generally as a result of the line being too light on the “dip stick”. Therefore, the urine screen would have to be sent to a lab for further testing.

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.