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
Extraction is a separation method that is often used in the laboratory to separate one or more components from a mixture. Sucrose was separated at the beginning because it is the most immiscible and it’s strongly insoluble. Next Acetylsalicylic Acid was separated which left Acetanilide alone. Variety steps could have led to errors occurring. For example the step of separation, when dichloromethane layer was supposed to be drained out, it could be possible some aqueous layer was drained with it. Which could make the end result not as accurate. Also errors could have occurred if possibly some dichloromethane was not drained out. Both way could interfere with end result of figuring the amount of each component in the mixture. The solids percentage were 22.1% more than the original. That suggests that solids weren’t separated completely which clarifies the reason the melting points that were recorded were a slightly lower than the actual component’s melting point. The melting point for Acetylsalicylic Acid is 136 C but that range that was recorded during the experiment was around 105 C to 118 C. The melting points were slightly lower than the literature value. Sucrose was the purest among all component due to its higher melting point which follows the chemical rule that the higher the melting point the more pure the component
Prodigiosin can formulate into a biopigment, henceforth contribute to the production of important medicinal materials. It is a highly desirable molecule, as of its characteristic traits being; antibacterial, antimycotic, antimalarial, antitumour and also immunosuppressant. (Prad...
Western blot has been a revolutionary technique for identifying the expression of proteins within relative molecular biological samples that shared the same ancestor. 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 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 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 lab is to learn how to properly conduct two different macromolecules test, the nucleic test and protein test in order to identify whether four different types of food, contain proteins and nucleic acid. The way an individual can determine if a specific macromolecule is present is by conducting qualitative tests, which allows an individual to determine whether a certain macromolecule is present by observing the color change. Additionally, for statistical analysis semi-quantitative tests will be conducted as well to determine the relative amount of a macromolecule that is present in the food based on the color change. (Dooley 20). Moreover, before conducting this experiment an individual must determine the positive and negative
Finally, the last part of the experiment examined the enzyme activity at different pH levels. Four sets of 11 tubes were set up in this part. The procedure for this part is the same as before, but 4 other buffers were substituted for the standard pH 7.3 phosphate buffer. Set A used the 5.5 pH buffer while set B used the 6.5 pH buffer. The buffer of pH 8.5 was used for set B and for set D the pH was 9. The absorbance readings for 4 sets were taken and recorded in table 13. Using the linear equation that the best-fit line gave for each set, the Km and the Vmax of each set were determined. Then, table 15 was made by dividing the Vmax by the Km. of the four pHs. The Vmax and Km of the control set were also used to make
In preparing for the quantitative test for the Bio-Rad protein assay, a spectrophotometer was switched on. Ten test tubes were used and that the known and unknown protein samples were tested duplicate. Tubes one and two were the 0.2 mg/ml protein, three and four 0.3 mg/ml protein, five and six 0.6 mg/ml protein, seven and eight 0.9 mg/ml protein, and lastly nine and ten
In this experiment, in the first part, the best concentration of enzyme was determined by recording the absorption over time. In the second part, the best concentration was selected from the previous experiment which was C and the optimum pH was determined.
Note: The revised version of the method (9.1.) differs in one major point from reference 9.4.: Preparation of samples and standards for analysis. It is now ...
The Vmax values, as determined from the Lineweaver-Burk plot, for the uninhibited, half uninhibited, and inhibited enzymes were, 0.3647, 0.1262, and 0.3087 μmol/min respectively. The non-linear regression V¬max¬ values for the same enzyme were 0.3343 (9.09% error as compared to Lineweaver-Burk plot), 0.1264 (0.16% error), and 0.2694 μmol/min (14.6% error) respectively. The differences in the values are due to the presence of error introduced by a Lineweaver-Burk plot, where data points at higher and lower substrate concentrations are weighed differently (Tymoczko, p.115). This error is the reason why a Michaelis-Menten plot is preferred.
The concentration of the following solutions can be measured using a spectrophotometer. In our lab, we will be using the Bradford Assay procedure to measure the concentration of our protein samples. A spectrophotometer is a machine that passes light through a cuvette that can detect how much light a solution can absorb (Absorbance). The absorbance from our measured unknowns can be used to determine the concentration of protein in the solution. If any of our test solutions produce the highest absorptivity, then that solution has the highest concentration of protein because more protein more protein molecules will bind to the Coomassie dye, yielding a dark blue color. If the brand company of muscle milk claims their product have more protein, then we expect to have a high absorptivity and dark blue color for Muscle Milk than the other
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.
This assay was used to measure the concentration of protein in a solution. Use it to determine the concentration of our saved supernatants 1-6. The total amount of protein is determined and so is the phosphatase activity. By combining the Bradford reagent with our diluted supernatants in a test tube and letting sit for 5 minutes to be read at 595nm we were able to determine the mg/ml of protein. The absorbance was read on a spectrophotometer which measures the light intensity. Using a standard curve we were able to get our readings. The procedure was taking the six supernatants and aligning them to six test tubes. A solution was created with 50x dilution for sup1, 10x for sup 2-3, and no dilution for sup 6. A Bradford reagent was then added to each. Afterwards analyzed using the A595 wavelength. A formula was then used to determine the amount of active protein. Sup 1 yielded 104.31 mg/ml, sup 2 yielded 22.33 mg/ml, sup 3 yielded 1.21 mg/ml, and sup 6 yielded 0.66
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.
Due to the nature of amino acids, a titration curve can be employed to identify