Assay

is also catalysed in the other direction by LDH during the Cori cycle. This reaction can be assayed using spectrophotometric techniques; peak absorbance of NADH is at 340nm, but the peak absorbance of NAD+ is 259nm (Powers, et al., 2007). Enzyme assays are performed to measure the rate of an enzyme-catalysed reaction, known as the enzyme activity. Many factors can affect the enzyme activity (including temperature, pH, substrate concentration), so all conditions apart from the one being…

P450-GloTM bioluminescent CYP assay Three concentrations of PTV (1, 10, 100 µM) were conducted to detect the luciferin PPXE activity against recombinant CYP3A4 and CYP3A5. A significant decrease in the luminescene activity in both recombinant CYP3A4 and CYP3A5 was observed in 10 µM and 100 µM of PTV (Fig 1). It seems that PTV is a candidate drug for metabolism against CYP3A4 and CYP3A5 enzymes. Metabolite profiling and Identification After incubating PTV with HLMs in the presence of a NADPH…

Malachite Green Assay: The reaction that AGPase catalyzes produces PPi. The resulting PPi that is produced is hydrolyzed by an inorganic pyrophosphatase (Sigma Aldrich) to generate phosphate (Pi). Each reaction for the assay was conducted on a 96 well plate and the reaction mixture was 50µL and contained Pre-Mix which contained 50mM HEPPS, pH=8.0 (MP Biomedicals), 10mM MgCl2, 1.5mM FBP (Sigma Aldrich), and 0.2mg/mL Bovine Serum Albumin (Sigma Aldrich), and 1.5mU/µL yeast inorganic…

a bond with the active form of trypsin in the matrix (Sugumaran). Two assays were performed. The first assay was to determine if there was any active trypsin by employing Benyzol-DL-arginie-p-nitroanilide (BAPNA), which forms an observable yellow color (from p-nitroaniline) when cleaved by active trypsin. The second assay used biorad protein assay reagent to estimate the concentration of the protein that was eluted. Both assays required a spectrometer to observe the color changes by absorbance.…

spectrophotometrical. The assay is a spectroscopy analytical technique based upon the absorbance maximum for an acidic solution of Coomassie Brilliant Blue. From the assay is observed the maximum absorbance for an acidic solution of Coomassie Brilliant Blue G-250 mostly shifts from 465 to 595 nm upon binding to protein studied occurs. Both ionic interaction and hydrophobic are used to stabilize the anionic form of the dye, which experience a visible change in the color (Stoscheck). This assay…

Homogenates of liver were provided. The homogenates were made by homogenizing liver in a 1:20 w/v (1g: 20 ml) ratio in a sucrose-phosphate buffer, in which 250mM of sucrose and 50mM NaPO, with a pH of 7.4 were added. This information was used to conduct an experiment to determine the concentration of DNA, protein, and carbohydrates in different homogenate samples, including heart, liver, and kidney of Bos taurus, while also looking for the diverse protein sizes found in each homogenate sample.…

failure of translation could be the limited knowledge that scientists working in biomarker discovery have in analytical, diagnostic and regulatory requirements for clinical assays (Fuzery et al., 2013). Over the last few decades a number of key FDA approved…

Oxaloacetate, found in the mitochondrion, is a four-carbon molecule. In this project we performed a Bradford Assay and an SDH (Succinate Dehydrogenase) Assay. The goal of this project was to work with enzymes to gain a better understanding of how enzymes function and how they are impacted with the introduction of a variety of factors. A significant part of the experiment was determining the protein concentration of the prepared mitochondrial fraction which will be further explained. The reason…

Toxoplasmosis is typically diagnosed using serological analysis. Multiple immunological assays can be used, including enzyme-linked immunosorbent assay (ELISA), indirect fluorescent antibody (IFA) technique, and immunosorbent agglutination assay (ISAGA). The Sabin-Feldman dye test, however, is the gold standard immunological assay. In the Sabin-Feldman dye test, the patient’s serum is treated with both live T. gondii organisms and methylene blue dye. If the patient has been previously…

Common tumors are often caused by mutations in the genes that may potentially lead to varying protein structures, which do not correlate with their proper functions (1). Majority of these mutations arise as a result of substituting one nucleotide base pair for another, but a significant number are also from adding or removing one of more of these nucleotides that ultimately disrupts protein translation (1). One of the mutated genes linked to cancer is BRCA1 (2,3,4). BRCA1 stops tumor growth by…