The purpose of today's lab work is to:
- 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
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).
Bradford 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:
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
- 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:
- 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
Figure 1: Raw Data: Fluorescence of alpha-Chymotrypsin Blanks as a Function of the Wavelength of Incident Light (nm)
The above figure is the raw data for the fluorescence of each of the alpha-chymotrypsin blanks as a function of the wavelength of incident light. Each curve on the graph represents a different concentration of alpha-chymotrypsin. The concentrations were converted from nM to mg/mL chymotrypsin.
Figure 2: Raw Data: Fluorescence of Lysozyme Samples Digested with alpha-Chymotrypsin as a Function of the Wavelength of Incident Light (nm)
The above figure is the raw data for the fluorescence of each of the lysozyme samples degraded with alpha-chymotrypsin as a function of the wavelength of incident light. Each curve on the graph represents a different concentration of lysozyme. The concentrations were converted from nM to mg/mL.
Figure 3: Fluorescence Lysozyme+Peptides as a Function of the Wavelength of Incident Light (nm)
The above figure shows the fluorescence of the lysozyme+peptides in the lysozyme samples as a function of the wavelength of incident light. In order to get this data, I made the following corrections to the raw data:
Figure 4: Fluorescence Intensity of Lysozyme+Peptides as a Function of the Concentration of the Lysozyme+Peptides
- I corrected the fluorescence of the lysozyme samples for instrumental noise by subtracting the fluorescence for the last .5nm measured from each of the fluorescence measurements for each respective sample
- I corrected the alpha-chymotrypsin blanks for instrumental noise in the same way that I corrected the lysozyme samples
- I then subtracted the corrected fluorescence values for the alpha-chymotrypsin blanks from those of the lysozyme samples for each respective wavelength and sample concentration. This resulting data was the fluorescence of just the lysozyme and peptides that were in the lysozyme sample. It effectively subtracted out any fluorescence that was from the alpha-chymotrypsin. This data is the data that the graph shows.
The figure above will be the calibration curve for all of our fluorescence measurements of the AuNP fibers. it shows the fluorescence intensity of the lysozyme+peptides as a function of the concentration of the lysozyme+peptides. In order to get this data, I did the following:
- I integrated the area under each of the curves for the corrected lysozyme fluorescence from 420-650nm
- I did the same thing for the corrected alpha-chymotrypsin fluorescence
- For each concentration, I subtracted integrated area of the alpha-chymotrypsin blanks from that of the lysozyme samples. This gave the fluorescence intensity of the lysozyme+peptides that were in the lysozyme samples.
- I then plotted concentration vs fluorescence intensity.
Figure 5: 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 subtracting the absorbance of a Bradford blank* from the absorbance each 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 6: 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 7: 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 5 and 6.
Figure 8: 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.
The fluorescence intensity of the lysozyme increased linearly with the concentration of alpha-chymotrypsin used to digest the lysozyme.
In our fluorescence assay, peptides were labeled so that they would fluoresce; as the concentration of peptide in the sample increased, the fluorescence would increase. alpha-Chymotrypsin degrades proteins, including lysozyme, which would cause the concentration of peptide in the sample to increase as the protein is degraded into peptides. Thus, since the fluorescence of the samples increased as the concentration of alpha-chymotrypsin increased, we can infer that increasing the concentration of the alpha-chymotrpysin increased the rate at which the lysozyme was degraded.
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 based on our Bradford Assay data.