Today's objective is to use a Bradford Assay to measure the absorbance of AuNP fibers that have been degraded by 1nM alpha-chymotrypsin for varying lengths of time.
- 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 58.59375µM.
- 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:
- 30 min
- 90 min
- 120 min
- 1440 min (24 hours)
- 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 1nM 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:
M1=concentration of alpha-chymotrypsin stock = 58.59375µM
V1=volume of protease stock needed
M2=concentration of alpha-chymotrypsin needed in the sample tube = 1nM = 0.001µM
V2=volume of the solution in the sample tube = 1mL
- Because the concentration of chymotrypsin was so low, we did a serial dilution or our chymotrypsin stock:
- First, we made a 1/400 dilution of our stock protease in Tris buffer. We pipetted 2.5µL of our stock solution into 997.5µL of Tris buffer.
- 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 1nM in each:
(0.0171µL initial chymotrypsin stock) x (dilution factor of 400) = 6.83µL diluted chymotrypsin
- 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 and alpha-chymotrypsin stock calculated in steps 18.104.22.168 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. (The Bradford dilution was 1 part Bradford reagent:3 parts Tris buffer). 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.
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.