User:Nicole Bonan/Notebook/Chem 571 Lab Notebook/2015/11/18

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Objective Today's objective is to use a Bradford Assay to measure the absorbance of AuNP fibers that have been degraded by 1µM alpha-chymotrypsin for varying lengths of time. Protocol We made a stock solution of our protease, alpha-chymotrypsin, using 50mM Tris/10mM CaCl2 buffer (pH=8) and one of the stock masses of alpha-chymotrypsin that we measured out earlier in the semester. The concentration of this solution was 74.21875µM. We prepared samples of AuNP fibers that Dr. Hartings prepared before class: We spun the samples down at 300 RPM for 10 minutes We pipetted the water off of the samples, being careful not to break andy fibers We labeled the samples based on the time that they would be incubated in the 37 degree Celsius water bath: 15min 30 min 45min 60min 90min 120 min NOTE: We made two of each of the samples so that we could average the absorbance values of both samples for each time interval Next, we determined how much of our protease stock we should add to each of the sample tubes in order to make the final concentration of alpha-chymotrypsin 1µM and the final volume of the sample 1mL: We did the following calculation to determine how much of the alpha-chymotrypsin stock we would need to add to each sample and blank to bring the final concentration of alpha-chymotrypsin to 1nM in each: Let M1=concentration of alpha-chymotrypsin stock = 74.21875µM V1=volume of protease stock needed M2=concentration of alpha-chymotrypsin needed in the sample tube = 1µM V2=volume of the solution in the sample tube = 1mL M1V1=M2V2 V1=(M2V2)/(M1) V1=((1µM)(1mL))/(74.21875µM) V1=0.01347mL=13.47µL After that, we determined and how much Tris/CaCl2 buffer to add to the sample to bring the volume up to 1mL by subtracting the volume of the protease we added from the final volume of the sample: 1000µL-13.47µL=987.5µL We then added the volume of alpha-chymotrypsin stock and buffer calculated in steps 2.4.1 and 2.5, respectively, to each of the sample tubes. Next, we prepared 10 blanks: We labeled each blank with an incubation time, as we did for the samples We put the same volume of buffer and alpha-chymotrypsin stockin each blank as we did in each of the samples We placed all the samples and blanks in the 37 degree Celsius water bath for their respective incubation times. We did not vortex the samples because we did not want to break the fibers. While each sample and blank was incubating, we pipetted 600µL of pre-mixed Bradford dilution into the cuvette for the samples and blanks with which we would take the UV-Vis measurements. We also pipetted 1650µL of buffer into each of the cuvettes that would be used for the samples and blanks. When the samples and blanks were finished incubating, we prepared them for measurement: We removed them from the water bath and spun the samples and blanks down at 12000 RPM for 1 minute We pipetted 750µL of each of the samples or blanks into their respective cuvettes We recorded the UV-Vis spectrum for the samples and blanks in the cuvettes from 400-800nm. Data Figure 1: Absorbance of alpha-Chymotrypsin Blanks as a Function of the Wavelength of Incident Light (nm) The above figure shows the absorbance of the alpha-chymotrypsin blanks as a function of the wavelength of incident light. The absorbance was corrected by first averaging the raw absorbance data for each duplicate sample and then subtracting the absorbance of a Bradford blank* from the absorbance of each averaged data point at their respective wavelengths. The absorbance was then corrected by subtracting the absorbance at the isosbestic point of each sample and blank from all of the absorbance values for the respective sample and blank. NOTE: The blank was just Bradford reagent and Tris buffer. Figure 2: Absorbance of AuNP Fiber Samples as a Function of the Wavelength of Incident Light (nm) The above figure shows the absorbance of the AuNP fiber samples as a function of the wavelength of incident light. The absorbance was corrected in the same way that the absorbance for the alpha-chymotrypsin blanks was corrected. Figure 3: Absorbance of alpha-Chymotrypsin Blanks and AuNP Fiber Samples at 600nm as a Function of Incubation Time (min) The above figure shows the absorbance of the alpha-chymotrypsin blanks and the AuNP fiber samples at 600nm as a function of the amount of time that they were incubated. The absorbance values are taken directly from the absorbances in Figures 1 and 2. Figure 4: Absorbance of AuNP Fiber Samples-alpha-Chymotrypsin Blanks at 600nm as a Function of Incubation Time (min) The above figure shows the absorbance of the AuNP fiber samples (after subtracting out the absorbance of the alpha-chymotrypsin blanks) at 600nm as a function of incubation time. It effectively shows the absorbance of the peptides and AuNP that had gone into solution as a result of degradation by alpha-chymotrypsin.