Finish taking fluorescence data from yesterday's lab
Use a Bradford Assay in order to measure alpha-chymotrypsin degradation as a function of incubation time
Protocol
Dr. Hartings's protocol for today is here. We are following his protocols for fluorescence and for the Bradford Assay.
Fluorescence Analysis of Protease Degradation
Yesterday, we measured the fluorescence of all of our samples of alpha-chymotrypsin + lysozyme. Today, we finished the fluorescence measurements for the alpha-chymotrypsin blanks as follows:
We had finished creating our blanks yesterday. Just before we measured the fluorescence of each blank, we combined the following 1.5mL Eppindorf tubes:
20uL of the blank to be measured
140uL of Assay Buffer
40uL of Assay Reagent
We then pipetted this mixture into a 1mL quartz cuvette and took fluorescence measurements (excitation wavelength was 390nm and emission wavelengths were 400 to 650nm).
Brasford Analysis of Protease Degradation
First, we made a stock solution of our protease, alpha-chymotrypsin, using Tris/CaCl2 buffer and one of the stock masses of alpha-chymotrypsin that we measured out earlier in the semester. The concentration of this solution was 40.625µM.
Next, we prepared 7 samples of AuNP fibers that Dr. Hartings prepared for us:
We spun the samples down at 1500 RPM for 1 minute
We pipetted the water off of the samples
We labeled the samples based on the time that they would be incubated in the 37 degree Celsius water bath:
2 hours
1.5 hours
1 hour
45 min
30 min
15 min
10 min
We measured the mass of each of the samples (the mass included the mass of the Eppindorff tube and the sample inside it)
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: Let M1=concentration of protease stock = 40.625µM V1=volume of protease stock needed M2=concentration of alpha-chymotrypsin in the sample tube = 1µM V2=volume of the solution in the sample tube = 1mL M1V1=M2V2 V1=(M2V2)/(M1) V1=((1µM)(1mL))/(40.625µM) V1=0.0246mL=24.6µL
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-24.6µL=975.4µL
We then added the volume of buffer calculated in step 2.6 to each of the sample tubes. We did not add the alpha-chymotrypsin stock; we waited to add this until just before we began each sample's incubation.
Next, we prepared 7 blanks:
We labeled each blank with an incubation time, as we did for the samples
We put the same volume of buffer in each blank as we did in each of the standards
Next, we added the volume of alpha-chymotrypsin that we calculated in step 2.5 to the 2 hour, 1.5 hour, and 1 hour samples and banks. We vortexed all of these solutions and placed them in the 37 degree Celsius water bath for 2 hours, 1.5 hours, and 1 hour, respectively.
We then added the volume of alpha-chymotrypsin calculated in step 2.5 to the 15 minute and 30 minute samples and blanks. We vortexed all of these solutions and placed them in the 37 degree Celsius water bath for 15 minutes and 30 minutes, respectively.
We then added the volume of alpha-chymotrypsin calculated in step 2.5 to the 45 minute sample and blank. We vortexed them and placed them in the 37 degree Celsius water bath for 45 minutes.
While we were waiting for the solutions to incubate, we pipetted 600µL of pre-mixed Bradford dilution into each of 14 cuvettes. These cuvettes would be the ones from which we took our UV-Vis measurements.
When the 15 minute sample and blank were finished incubating, we prepared them for measurement:
We removed them from the water bath and spun the sample (but not the blank) down at 300 RPM for 1 minute
We pipetted 1650µL of buffer into each of the cuvettes that would be used for the 15 min sample and the 15 min blank
We pipetted 750µL of the 15 min sample into the 15 min cuvette. We did the same for the 15 min blank.
Next, we recorded the UV-Vis spectrum for the two cuvettes from 400-800nm.
After that, we repeated steps 8-9 for the 30 minute, 45 minute, and 1 hour samples and blanks.
We then repeated step 5-6 for the 10min sample and blank
We repeated steps 8-9 for the 1.5 hour sample and blank
We repeated steps 8-9 for the 10 min sample and blank
We repeated steps 8-9 for the 2 hour sample and blank
Data and Analysis
Fluorescence
Figure 1: Fluorescence of Lysozyme in Varying Concentrations of alpha-Chymotrypsin (nM) as a Function of the Wavelength of Incident Light (nm)
The above figure shows the fluorescence of lysozyme in samples of varying concentrations of alpha-chymotrypsin as a function of the wavelength of incident light. The fluorescence of each sample was corrected for the blanks by subtracting the fluorescence of the blank from the fluorescence of the sample for every wavelength measured. Each curve on the graph represents a different concentration of alpha-chymotrypsin.
Figure 2: Fluorescence Intensity of Lysozyme as a Function of the Concentration of alpha-Chymotrypsin
The above image shows the fluorescence intensity of lysozyme as a function of the concentration of alpha-chymotrypsin that it is reacted with.
Bradford Assay
Figure 3: Absorbance of the Blank as a Function of the Wavelength of Incident Light (nm)
The figure above shows the absorbance of the blank as a function of the wavelength of incident light. The blank is the same blank that was made and measured in the protocol from September 23. This blank consisted of the Bradford Assay Reagent, Bradford Assay Buffer, and 50mM Tris + 50mM NaCl buffer. There was no protease, protein, or AuNP in this blank.
Figure 4: Absorbance of the alpha-Chymotrypsin Blanks as a Function of the Wavelength of Incident Light (nm)
The figure above shows the absorbance of the alpha-chymotrypsin blanks as a function of the wavelength of incident light. Each colored curve represents a blank that was incubated for a specific amount of time as indicated in the legend. These values are not corrected; they are the raw data.
Figure 5: Absorbance of the AuNP Fiber Samples as a Function of the Wavelength of Incident Light (nm)
The figure above shows the absorbance of the AuNP fiber samples as a function of the wavelength of incident light. Each colored curve represents a sample that was incubated for a specific amount of time as indicated in the legend. These values are not corrected; they are the raw data.
Figure 6: Absorbance of the alpha-Chymotrypsin Blanks, corrected for the Blank, as a Function of the Wavelength of Incident Light (nm)
The figure above shows the absorbance of the alpha-chymotrypsin blanks as a function of the wavelength of incident light. The absorbance values for each of the alpha-chymotrypsin blanks were corrected by subtracting the absorbance value of the blank from the absorbance value of the alpha-chymotrypsin blanks for every wavelength. Each colored curve represents an alpha-chymotrypsin blank that was incubated for a specific amount of time as indicated in the legend.
Figure 7: Absorbance of the AuNP Fiber Samples, Corrected for the Blank, as a Function of the Wavelength of Incident Light (nm)
The figure above shows the absorbance of the AuNP fiber samples as a function of the wavelength of incident light. The absorbance values for each of the samples were corrected by subtracting the absorbance value of the blank from the absorbance value of the samples for every wavelength. Each colored curve represents a sample that was incubated for a specific amount of time as indicated in the legend.
Figure 8: Absorbance of alpha-Chymotrypsin Blanks, Corrected for the Blank and Isosbestic Point, as a Function of the Wavelength of Incident Light (nm)
The figure above shows the absorbance of each of the alpha-chympotrypsin blanks as a function of the wavelength of incident light. The absorbance was first corrected for the blank by subtracting the absorbance of the blank at each wavelength from the absorbance of the alpha-chymotrypsin blank at each wavelength. This procedure was done for every alpha-chymotrypsin blank. The absorbance was then corrected for the isosbestic point. Since the isosbestic point was at 535nm, and the absorbance at 535nm was not 0 after correcting for the blank, the absorbance was corrected again in order to make the isosbestic point have an absorbance of 0. Thus, the absorbance of each alpha-chymotrypsin blank at 535nm was subtracted from every absorbance measurement for that alpha-chymotrypsin blank.
Figure 9: Absorbance of the AuNP Fiber Samples, Corrected for the Blank and Isosbestic Point, as a Function of the Wavelength of Incident Light (nm)
The figure above shows the absorbance of each of the AuNP fiber samples as a function of the wavelength of incident light. The absorbance was first corrected for the blank by subtracting the absorbance of the blank at each wavelength from the absorbance of the sample at each wavelength. This procedure was done for every sample. The absorbance was then corrected for the isosbestic point in the same way that it was corrected for the alpha-chymotrypsin blanks (see Figure 8).
Figure 10: Calibration Curve for the Bradford Assay
The figure above is the calibration curve for both the alpha-chymotrypsin blanks and the AuNP fiber samples. It shows the absorbance of the blanks and samples as a function of incubation time at a wavelength of incident light of 600nm.
Conclusions
Fluorescence Assay
Bradford Assay
Degrading AuNP fibers with alpha-chymotrypsin should have caused the fibers to degrade into peptides. By using a Bradford Assay, we effectively labeled peptides so that they would absorb light between 500-700nm. Thus, when we measured absorbance, the samples with more peptide would have a greater absorbance than the samples with less peptide.
Theoretically, the samples with more peptide should have been the samples that had incubated longer and thus had been able to digest more AuNP fibers. By making samples that had incubated for varying lengths of time and then measuring their absorbance, our goal was to determine the rate at which our protease degraded the AuNP fibers.
Unfortunately, our calibration curve does not provide much useful data about the absorbance of our samples as a function of time. We cannot really determine how fast the alpha-chymotrypsin degrades the AuNP fibers.
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