Defensin (DF) is a newer cytolytic antimicrobial peptide having molecular weight of 4 kDa having antimicrobial activity against wide range of gram positive and gram negative bacteria. The present work describes method development of recombinant Defensin protein using internal standard Bovine serum albumin (BSA). A simple, precise and accurate reverse phase high performance liquid chromatography method has been developed and validated for quantification of Defensin protein using BSA as an internal standard using Enable Q C18 column (250mm x 4.6 mm I.D, 5μm particle size 300ºA) as column, water: acetonitrile (60:40 v/v) with Trifluoroacetic acid 0.1% as mobile phase, flow rate of 1ml/min and detection was carried out at 280 nm. The retention time of BSA and Defensin was 2.342 and 5.279 min respectively. The linearity range was found to be 10-50 μg/ml, co-relation coefficient was found to be 0.999. The limits of detection and quantitation of the method were 1.010 and 3.062 μg/ml respectively. So, the developed method was accurate, precise and robust.
Key words: Recombinant Defensin, BSA, RP-HPLC, validation.
Human Defensins (1-5) are small, cationically charged, cysteine-rich endogenous antibiotic peptides with antimicrobial and cytotoxic properties that contain 29-35 amino acid residues, including six invariant disulphide linked cysteines moiety having a molecular weight of 4-5 kDa. The novel trend in drug development is the design and development of biopharmaceuticals, thus, the pharmaceutical industry has focused on bio molecules method development and validation (8-9). HPLC is a widely used technique for proteins and peptides because of its ease of usage, higher selectivity and faster analysis. In this study one of the antim...
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...Table 2). The results for intraday (0.30 – 0.80%) & interday precision (0.34 – 0.62%) study performed on 3 concentrations (20, 30 & 40 μg/ml) of standard solution of BSA & Defensin indicate that the purported method being precise (Table 2). The values for LOD & LOQ were found to be 1.010 & 3.062 μg/ ml respectively (Table 2). These data suggested that the proposed method was sensitive for the estimation of both the proteins.
The recovery study was performed by the standard addition method. The amount of Defensin was calculated by putting value of ratio of Defensin to BSA (IS) in regression equation. And % recovery was calculated for Defensin. The mean recovery obtained was 99.82 – 101.85% which was within the limit of 98 – 102% which proves that the method was accurate (Table 3). To evaluate robustness of the developed method, few parameters were deliberately varied
Data from Table 1. confirms the theory that as the concentration of glucose increases so will the absorbance of the solution when examined with the glucose oxidase/horseradish peroxidase assay. Glucose within the context of this assay is determined by the amount of ferricyanide, determined by absornace, which is produced in a one to one ratio.1 Furthermore when examining the glucose standards, a linear calibration curve was able to be produced (shown as Figure 1). Noted the R2 value of the y = 1.808x - 0.0125 trend line is 0.9958, which is statistically considered linear. From this calibration curve the absorbance values of unknowns samples can be compared, and the correlated glucose concentration can then be approximated.
Moreover, the class average curve shows a similar trend, as the curve flattens, at 70% but with an enzyme activity of 5.3 x10-3 seconds. This indicates that even though the saturation point is the same it was considerably lower than our results, which could indicate sources of systematic error in the design of the practical.
Moreover, the sensitivity and specificity of the western blot (Immunoblotting) enables it a common technique for determining specific protein levels in clinical samples. Since the antibody is specific to the antigen immunospecificity it enables the target protein to be identified. Western blotting can produce quantitative data about that protein, which in this case shows the difference between bands in each of the protein samples. The western blot is an analytical technique used to detect specific proteins in the given sample of tissue homogenate or extract. The proteins are then transferred to a membrane (in this case, nitrocellulose), where they are stained with antibodies specific to the target protein [1] [2].
The purpose of this experiment was to discover the specificity of the enzyme lactase to a spec...
Cardiovascular Diseases (CVD) are the currently the leading cause of death globally for both men and women accounting for 21.9 per cent of total deaths and is projected to increase to 26.3 per cent by 2030 . Statins are the treatment of choice for the primary and secondary prevention of cardiovascular disease and in the management of hypercholesterolaemia because of their proven efficacy and safety profile. Evidences are showing their effectiveness in reduction of cholesterol synthesis and number of pleiotropic effects, which may be cholesterol dependent and cholesterol independent. The present review focus on the origin, properties and effects of statins on endothelial function ( non lipid action of statins) through the increase of endogenous production of NO in different pathways.
The actual amount of crude product was determined to be 3.11 grams. The percent yield of the crude product was determined to be 67.75 %. The actual amount of pure product formed was found to be 4.38 grams. The percent yield of the pure product was determined to be 95.42%. Regarding the thin layer chromatography, the line from the solvent front is 8 centimeters.
Due to the nature of amino acids, a titration curve can be employed to identify
The Standing Committee of the Institute of Medicine used a broad range of research and studies in order to ensure upmost accuracy when compiling the DRI chapter for niacin. One reference, Grace A. Goldsmith M.D., presents information useful for setting the niacin DRI in her article “Niacin-Tryptophan Relationship in Man and Niacin Requirements.”
Compounding all of these solutions, the pharmaceutical industry needs to conduct extensive research on developing new antibiotics for various pathogenic bacteria by studying the bacterial structure. This will help scientists to formulate ways of counteracting the functions of the various constituents of bacteria.
The purpose of this experiment was to learn and preform an acid-base extraction technique to separate organic compounds successfully and obtaining amounts of each component in the mixture. In this experiment, the separation will be done by separatory funnel preforming on two liquids that are immiscible from two layers when added together. The individual components of Phensuprin (Acetylsalicylic acid, Acetanilide, and Sucrose as a filler) was separated based upon their solubility and reactivity, and the amount of each component in the mixture was obtained. Also, the purity of each component will be determined by the melting point of the component.
In undertaking the experiment, the hypothesis “if the number of Alka Seltzer tablets reacted increases, then the maximum rate of reaction will increase,” was formed. When graphing the relationship between the maximum rate of reaction and the number of Alka Seltzer tablets reacting, Graph 7 produced a line of best fit with a constant increasing slope that passed through the origin (0,0). This is characteristic of linear graphs, which have the general equation, y=mx, where m is the slope, a constant term, and y and x are changing variables that are directly proportional (i.e. y ∝x). Hence, it can be deduced that Graph 7 is a linear graph, and that there is a linear relationship between the maximum rate of reaction and the number of Alka Seltzer tablets, where they are directly proportional. That is, as the number of Alka Seltzer tablets increased, the maximum rate of reaction increased, supporting the hypothesis. As the true value of the maximum rate of reaction per Alka Seltzer tablet was not known, and a value for comparison was unavailable, the accuracy of the results could not be determined. However, due to the scatter in Graphs 2 to 7, it was evident that the results had low precision. In future, repeating the experiment using different and/or new apparatus will aid in detecting systematic errors and improve the accuracy and validity of the results.
For the lab experiment for Membrane Damage, we tested the extract pigment and diluted it. When the pH solutions are added, this will cause it to be in a range of absorbance. We used materials as follows. Obtaining a beet we proceeded to cut small individual cubes. We then rinse each cube to remove any damaged pigments with deionized water. Using a blender, we blend the beets with 15 mL of pH 7 DI water. After blending we used cheesecloth to separate the liquid from the solids for easier centrifuge process. Then we put the liquid beet into a centrifuge tube and centrifuge it for 5 minutes at 2500 rpm. We then remove the supernatant into a beaker, and discarded the sediment. Using a 1:4 ratio mixture of the supernatant and deionized water, we made a stock solution. We then tested the stock solution’s absorbance with a spectrophotometer, and place 1 mL of the solution into separate test tubes. Next we added an additional 4 mL of pH solutions in the 2-11 range into each test tube. After mixing, we tested the absorbance for each solution using a spectrophotometer.
The chemical structure of doripenem is similar to other carbapenem antibiotic. It compromised a beta lactam ring fused with 5-membered ring. Differ with penicillin, at carbon 1, doripenem has a beta methyl group which prevent hydrolysis of DHP-1. Among carbapenem members, only imipenem does not exert this physical feature. Therefore, imipenem need an additional of dehydropeptidase-1 inhibitor to sustain the microbial activity. Oth...
= Before conducting the experiment I would conduct a simple test for the protein by placing a sample of the albumen into a test tube and add biurett reagent. This contains copper (II) sulphate and sodium hydroxide.
One of the greatest achievements in the 20th century was the invention and mass production of antibiotics. Antibiotics or antimicrobials are chemical drugs that are used to treat a variety of different infectious bacterial diseases by destroying or slowing the growth of the bacteria. Antibiotics are toxic to the target cell but do not harm the host. They are designed to attack various kinds of parasites, fungus, and bacteria. Contrary to popular belief antibiotics do not work against viruses like those that cause the common cold. Antibiotics are broken down into different classifications because antibiotics are selective killers, which mean that it has been manufactured to kill a certain malicious bacteria strain. Each antimicrobial differs from one another by their physical and chemical properties and by their antibacterial spectrum of activity. The different kinds of antibiotics fight off the infection in different ways. The different ways the antibiotic functions is by inhibiting the bacterial cell from metabolizing, by preventing the bacteria’s cell wall synthesizing, by creating a permeable membrane of the bacteria cell, or by suppressing nucleic acid transcription and replication. Most antibiotics today are semisynthetic, which means their natural derivative has been altered. Antibiotics have two distinct groups that was separated by their effect on bacteria. Bactericidal antimicrobials kills bacteria while bacteriostatic agents hinder the bacteria from growing or reproducing. They are also seperated depending on their activity sepectrum. Narrow antibiotics target a spesfic group of bacteria while a broad spectrum targets many different kinds of cells.