13 Di Data Analysis 1 Calculate The Concentration Of Lysozyme Activity That Is Present In The Ewp And The Fractions E 1 (90.4 KiB) Viewed 93 times
13 Di Data Analysis 1 Calculate The Concentration Of Lysozyme Activity That Is Present In The Ewp And The Fractions E 2 (22.63 KiB) Viewed 93 times
13 Di Data Analysis 1 Calculate The Concentration Of Lysozyme Activity That Is Present In The Ewp And The Fractions E 3 (16.01 KiB) Viewed 93 times
13. Di Data analysis 1. Calculate the concentration of lysozyme activity that is present in the EWP and the fractions. Express this activity in units of abs/min/mL. Understandably, students often find calculating concentration of activity perplexing as it is a very unfamiliar unit. The best way to simply the value that comes from the spectrophotometer reading. It is the mL portion of the approach and think about the calculation is to proceed in the following manner: the abs/min is throughout the cuvette, but all of it originates in the volume of enzyme-containing solution that required units that confuses students, but remember, the measured activity is distributed you added to the cuvette-that is where the per volume" portion of the correct answer and the overall unit arises. When you do the calculation for the EWP, remember that you performed the assay using the diluted EWP as a source of enzyme, so you need to take that dilution into account also. 3. Calculate the total amount lysozyme activity that was present in the EWP that was loaded to the top of the column. Also calculate the total amount of lysozyme activity that was collected for fractions A, C, and E. Total amount of lysozyme activity is simply the concentration of activity multiplied by the volume added to the column (if we were talking about the EWP) or the volume collected if we are calculating this for a fraction. Calculate the % recovery of lysozyme activity for these fractions. You can calculate this because you know from calculation 2 how much lysozyme activity you loaded on the column, and you also know how much lysozyme activity was recovered in each fraction. The % recovery represents the percent fraction of what you loaded on the column that was recovered in any particular fraction. Please note that you do not calculate this for the EWP because you did not collect anything for that—that is what you loaded on the column. Calculate the concentration of protein in the EWP and each of the fractions. For this calculation you will require a calibration curve for the Bradford Assay. Normally, you would prepare a Bradford calibration curve at the same time you are performing assays on the cuvettes, but to save time, reagents and cuvettes, the data required to prepare your calibration curve will be posted on UM Learn. For preparing your calibration curve plot the ratio of the absorbances at 595/466 against the concentration of protein in ug/mL. Also calculate the total amount of protein 4.
Dilute EWP Sample name Abs @ 466 nm Abs @ 595 nm 0.257 0.465 0.628 0.607 C 0.260 0.278 E 0.281 0.286 llowing your 466/595 measurement Dilute A
Perform lysozyme assays on fractions C and E in the same manner as you did for fraction Record your results in the table below. Sample Name M. lysodeikticus cell wall hydrolysis (Abs/min) -0.017 Dilute EWP A с E 0.090
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