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Question: paraphrase.Part A For the wash buffer, the required mass of glycine was calculated and added to about 70% of the final volume of distilled water. After proper dissolution, the solution was titrated with a compatible acid to achieve pH 10. Then, the volume was adjusted to the correct level with distilled water.For the elution buffer, sodium chloride was first added to the desired concentration, followed by the calculated amount of glycine. Again, these were dissolved in approximately 70% of the final volume of distilled water. After titration to pH 10, the volume was adjusted to 50 ml with distilled water.For the 0.1 M sodium phosphate buffer with a pH of 6.24, the necessary calculations were carried out to determine the mass of sodium phosphate required. Following the procedure, the sodium phosphate was dissolved in about 70% of the final volume of distilled water. Then, the solution was titrated to the target pH and the volume was adjusted to 100 ml with distilled water. Part B The most essential principle to remember was to keep all samples COLD at all times, so ice was placed in a container in which the samples were to be placed.10 ml of pre-swelled activated CM-Sephadex was obtained and placed in a CLEAN 15 ml centrifuge tube. It was gently swirled, and any broken beads of gel found in the buffer above the gel were poured off. More glycine pH 10 buffer was added to the gel and gently stirred.An egg was obtained, and the white was separated from the yolk. The volume of the egg white obtained was measured and recorded. The egg white was then filtered through a double layer of cheesecloth by gently pressing the cheesecloth over the rim of a 100 or 150 ml beaker, which was placed in an ice bath.Each group transferred 3 ml of the egg filtrate to a clean 15 ml centrifuge tube and diluted it 1:5 (15 ml total volume) with glycine (pH 10) buffer. The volume was measured, and an A280 reading was taken. Two 1.5 ml samples were saved and labeled as Sample C (crude) and stored on ice.The clear buffer layer of the 10 ml of pre-swelled CM-Sephadex medium was removed. The filtered egg white was added to the CM-Sephadex, divided into two equal samples (approximately 12.5 ml per tube), and gently swirled for 10 minutes. The mixture was then centrifuged at approximately 500 rpm for 5 minutes.The supernatant was removed, and its volume was recorded. Its absorbance at A280 was measured using quartz cuvettes. Two 1.5 ml samples were taken from the supernatant fraction, labeled as W1 (wash 1), and placed on ice. The rest of the supernatant was discarded, but the CM-Sephadex was kept.15 ml of wash buffer (glycine pH 10 buffer) was added to the resin, gently swirled, and centrifuged as before to wash off any unbound proteins. The volume of the wash was measured, and its A280 was recorded. Steps 12 and 13 were repeated until the supernatant reading was near 0 at A280. A sample of this last wash (2 x 1.5 ml) labeled as W2 was saved on ice.10 ml of wash buffer was added to the CM-Sephadex, and the CM-Sephadex was gently poured down the side of a glass-stirring rod into the column. Buffer was allowed to slowly move out of the column below, and all the CM-Sephadex was ensured to fit into the column. The column was not allowed to run dry.Once the column was packed, any buffer from above the resin was removed, and 20 ml of elution buffer (0.5 M NaCl, 0.1M Glycine buffer pH 10) was added. 5 ml elution (EL) fractions were collected and placed on ice. Elution was continued until A280 came down to 0, adding more elution buffer if necessary.The high protein containing fractions were pooled together, and the volume was measured. Two 1.5 ml aliquots were kept on ice, labeled as the EL fraction.All centrifuge tubes (C, W1, W2, and EL) were placed in the freezer for next week’s practical.For results, a chromatography was needed, which is a graph of absorbance readings of all samples collected. The positions of C, W1, W2, and EL were indicated on the graph. Part C A standard curve was constructed from duplicate assays between 0 and 0.5 mg/ml of BSA protein. An individual standard curve was prepared using BSA and distilled water to ensure 2 ml of protein solution per assay.The standard curve was performed simultaneously with the protein sample analysis using the following method. It was noted that C, W1, W2, and EL samples needed to be diluted 100x before conducting the assay. Each sample was measured in duplicate. For each protein assay, 1 ml of the diluted sample was required. Reagent A (0.9 ml) was added to 1 ml of protein solution, including standard curve BSA dilutions as well as C, W1-2, and EL samples. The mixture was heated at 50 ºC for 10 minutes. Subsequently, the solutions were cooled in running tap water to room temperature, and then reagent B (0.1 ml) was added. The solutions were allowed to stand at room temperature for another 10 minutes.Following this, reagent C (3 ml) was added with immediate thorough mixing to ensure complete mixing within 1 second. The solutions were heated again at 50 ºC for 10 minutes. After cooling, the absorbance was read at 650 nm.All values were recorded neatly in a tabular form, and protein concentration for each sample was calculated from the standard curve, taking into account the dilutions. Part D Using 0.1 M Na phosphate (pH 6.24) buffer, a cell wall suspension was prepared at a concentration of 0.2 mg of cell wall per ml of phosphate buffer. This suspension was provided.To begin the experiment, 7 test tubes were labeled as Blank (B), C, W1, W2, EL, Ctrl, and commercial lysozyme. Each test tube contained 2.8 ml of the cell wall suspension and 0.2 ml of buffer. The absorbance reading for each tube was recorded after preparing.The spectrophotometer was set to 450 nm, and a blank reading was taken using plain phosphate buffer.0.2 ml of lysozyme-containing samples (C, W1, W2, EL, Ctrl, and commercial lysozyme) was added to each tube, and the tubes were quickly covered with Parafilm and gently mixed. Timing was initiated as soon as the lysozyme was added.The tubes were then placed in the spectrophotometer, and absorbance readings were recorded at 5 to 10-second intervals for approximately 2 minutes, extending the time if the absorbance was still decreasing.All values were recorded neatly in a tabular form.The Abs450 versus time in minutes was plotted, and the slope (Abs450 change per minute) was determined, correcting for any blank rate if present.One unit of lysozyme activity was defined as the amount of lysozyme that produces a 0.001 Abs450 change per minute.Finally, the enzyme activity (Units/ml) was calculated based on the determined slope and the definition of one unit of lysozyme activity.
Part A We started the wash buffer by determining and adding the appropriate quantity of glycine into around 70% of the ultimate volume of distilled water. The mixture was then dissolved thoroughly and titrated with a fitting acid until it reached pH 10. Finally, the volume was perfected with more distilled water. For the elution buffer, we first introduced sodium chloride to the needed concentration, and followed by adding the calculated amount of glycine. Both were dissolved in about 70% of the eventual volume of distilled water. After titration to pH 10, the solution volume was made up to 50 ml using distilled water. In preparation for the 0.1 M sodium phosphate buffer having a pH of 6.24, we conducted necessary calculations to determine the required amount of sodium phosphate to add. We then went ahead to dissolve the sodium phosphate in roughly 70% of the total volume of distilled water. Afterwards, we titrated the solution until we reached the target pH and fine-tuned the volume to 100 ml with distilled water. Part B It was crucial to ensure that all samples remained COLD at all times, hence we put ice in a container to house the samples. We then sourced for 10 ml of pre-soaked activated CM-Sephadex, put it in a CLEAN 15 ml centrifuge tube, and gently swirled it. Broken gel beads were located and poured off the top buffer layer. More glycine pH 10 buffer successfully stirred into the gel. An egg was procured, and its whites were separated from the yolk. We measured and documented the egg white volume before running it through a cheesecloth, fitted over a 100 or 150 ml beaker contained in an ice bath. Each group transferred 3 ml of the filtered egg white to a 15 ml clean centrifuge tube and diluted it to a volume of 15 ml with the glycine (pH 10) buffer. After measuring the volume, an A280 reading was taken. We then reserved two 1.5 ml samples, labeled them as Sample C (crude), and kept them on ice. We then removed the clear buffer layer off the 10 ml of prepared CM-Sephadex medium and added in the filtered egg white. The CM-Sephadex-medium-egg-white mixture was then divided into two samples (roughly 12.5 ml per tube), and swirled gently for 10 minutes. The mixture was then spun at about 500 rpm for 5 minutes in a centrifuge. The volume of the supernatant was noted and its absorbance at A280 was gauged using quartz cuvettes. Two 1.5 ml samples were extracted from the supernatant fraction, labeled as W1 (wash 1), then placed on ice. We kept the CM-Sephadex but discarded the remaining supernatant. We added 15 ml of wash buffer (glycine pH 10 buffer) to the resin, swirled gently, and centrifuged akin to the previous step, in order to rinse off any residual proteins. We gauged the volumes of the various washes and recorded their A280 values. We executed this step until we attained an A280 reading near 0 for the supernatant. We isolated a sample of the final wash (2 x 1.5 ml), labeled it W2, and placed it on ice. We then added 10 ml of wash buffer to the CM-Sephadex, and carefully poured it onto a glass stirring rod into a column. This allowed for the buffer to flow out slowly and position all the CM-Sephadex inside the column. It was key to ensure the column did not run dry. Once we packed the column, we cleared buffer from above the resin and incorporated 20 ml of elution buffer (0.5 M NaCl, 0.1M Glycine buffer pH 10). We collected the elution fractions (each 5 ml) and placed them on ice. We persisted with this elution until A280 reduced to 0, adding more elution buffer as needed. The fractions containing high protein levels were amalgamated, and their total volume was measured. We additionally held back two aliquots of 1.5 ml each, labeled as the EL fraction, and stored them on ice. We then proceeded to freeze all centrifuge tubes (C, W1, W2, and EL) in readiness for the ensuing week’s practical. To record our results, we developed a chromatogram, illustrating absorbance readings of all collected samples, while pointing out the positions of C, W1, W2, and EL. Part C We generated a standard curve through doubling assays between 0 and 0.5 mg/ml of BSA protein. We made sure that there were 2 ml of protein solution per assay by using BSA and distilled water. The process for the standard curve happened alongside the analysis of the protein sample and it was essential to thin down C, W1, W2, and EL samples by a factor of 100 prior to implementing the assay. We doubled every sample measurement. For every protein assay, we needed 1 ml of the diluted sample. We then added 0.9 ml of Reagent A to 1 ml of protein solution. This mix was inclusive of standard curve BSA dilutions as well as samples C, W1-2, and EL. We then maintained the mixture at 50 ºC for 10 minutes. The mixtures were then brought down to room temperature by cooling it under a running tap, after which we added 0.1 ml of reagent B. The solutions were left standing at room temperature for another 10 minutes. We then added 3 ml of reagent C with thorough and instant mixing. After collecting every measurement at one-second intervals for complete mixing, we again heated the solutions at 50 ºC for 10 minutes. After they cooled down, we read the absorbance at 650 nm. All measurements were captured neatly in table form and we calculated the protein concentration from each sample from the standard curve, considering the dilution factor. Part D We prepared a cell wall suspension using 0.1 M Na phosphate buffer (pH 6.24), with a concentration of 0.2 mg of cell wall per ml of phosphate buffer. The resulting suspension was then made available to us. In the beginning of the experiment, we labeled 7 test tubes as Blank (B), C, W1, W2, EL, Ctrl, and commercial lysozyme. Each tube received 2.8 ml of cell wall suspension and 0.2 ml of buffer, and we then measured absorbance for each tube. We set the spectrophotometer to 450 nm, and took a blank reading using plain phosphate buffer. We then introduced 0.2 ml of lysozyme-containing samples (C, W1, W2, EL, Ctrl, and commercial lysozyme) to each tube, quickly sealed them with Parafilm, and gentle mixed them. As soon as the lysozyme was added, timing commenced. The tubes were then placed in the spectrophotometer, and absorbance readings were taken at intervals of 5 to 10 seconds for roughly two minutes. We extended the time if the absorbance readings were still dropping. Every measurement was neatly captured in table form. We then drafted a graph of Abs450 against time (in minutes) and determined the slope (Abs450 change per minute). We made corrections for any blank rate if present. One unit of lysozyme activity was defined as the amount of lysozyme which induced a 0.001 Abs450 change per minute. With this in mind, we then computed the enzyme activity (Units/ml) using the slope values and definition of one lysozyme unit.
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