Today's objective is to use a Bradford Assay to measure the absorbance of AuNP Fibers degraded with 10nM alpha-chymotrypsin. Our goal is to measure the rate at which alpha-chymotrypsin degrades the fibers at a concentration of 10nM.
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 43.539µM.
We prepared 10 samples of AuNP fibers that Dr. Hartings prepared for us on October 12:
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:
168 hours (1 week)
48 hours
24 hours
2 hours
1.5 hours
1 hour
45 min
30 min
15 min
10 min
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 10nM and the final volume of the sample 1mL.
Because the concentration of chymotrypsin was so low, we did a serial dilution or our chymotrypsin stock:
First, we made a 1:20 dilution of our stock protease in Tris buffer. We pipetted 50µL of our stock solution into 950µL of Tris buffer. This brought the final concentration of the chymotrypsin to 2.177µM.
Next, we did the following calculation to determine how much of this solution we would need to add to each sample and blank to bring the final concentration of chymotrypsin to 10nM in each: Let M1=concentration of protease stock = 2.177µM V1=volume of protease stock needed M2=concentration of alpha-chymotrypsin in the sample tube = 10nM = 0.010µM V2=volume of the solution in the sample tube = 1mL M1V1=M2V2 V1=(M2V2)/(M1) V1=((0.010µM)(1mL))/(2.177µM) V1=0.00459mL=4.59µL
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-4.59µL=995.41µL
We then added the volume of buffer calculated in step 2.5 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 10 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 samples
Next, we added the volume of alpha-chymotrypsin that we calculated in step 2.4.1.2 to all of the samples and blanks except for the 10min sample and blank. We placed them all in the 37 degree Celsius water bath for their respective incubation times. We did not vortex the samples this time 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.
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 1650µL of buffer into each of the cuvettes that would be used for the samples and blanks
We pipetted 750µL of each of the samples or blanks into their respective cuvettes
Next, we recorded the UV-Vis spectrum for the samples and blanks in the cuvettes from 400-800nm.
Between the measurements for the 1 hour and 1.5 hour samples and blanks, we repeated steps 4-7 for the 10min sample and blank.
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 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 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.
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