User:Nicole Bonan/Notebook/Chem 571 Lab Notebook/2015/11/10

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Objective

Today's objective is to measure the fluorescence of AuNP fibers degraded with 1nM alpha-chymotrypsin for varying lengths of time.

Protocol

  1. We made a 1mL stock solution of our protease, alpha-chymotrypsin, by adding 1mL of 50mM phosphate buffer (pH=8) to one of the stock masses of alpha-chymotrypsin that we weighed out earlier in the semester. The final molarity of this sample was 51.172µM.
  2. 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:

    1. Let
      M1=concentration of protease stock = 51.172µM
      V1=volume of protease stock needed
      M2=concentration of alpha-chymotrypsin in the sample tube = 1nM = 0.001µM
      V2=volume of the solution in the sample tube = 1mL
      M1V1=M2V2
      V1=(M2V2)/(M1)
      V1=((0.001µM)(1mL))/(51.172µM)
      V1=0.00001954mL=0.01954µL
    2. Because the amount of chymotrypsin needed was so low, we did a serial dilution or our chymotrypsin stock:
      1. First, we made a 1:200 dilution of our stock protease in phosphate buffer. We pipetted 5µL of our stock solution into 995µL of phosphate buffer.
      2. 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:
        (0.01954µL initial chymotrypsin stock) x (dilution factor of 200) = 3.91µL diluted chymotrypsin
  3. We then determined the volume of phosphate buffer needed to bring the samples and blanks up to a total volume of 1000µL:
    1000µL-3.91µL = 996.1µL
  4. We prepared AuNP fiber samples:
    1. We used the samples that Dr. Hartings synthesized. We had seven AuNP samples total, one for each incubation time that we were interested in:
      1. 30min
      2. 60min
      3. 90min
      4. 120min
      5. 24 hours
      6. 48 hours
    2. We spun all the fiber samples down at 300 RPM for 10 minutes and pipetted off as much supernatant as we could.
  5. We added the volumes of diluted alpha-chymotrypsin and phosphate buffer that we calculated in step 2 and 3 to each of the samples and blanks. We put all of the samples and blanks into the 37 degree Celsius water bath for their respective incubation times. We were careful not to disrupt the AuNP fiber pellet as we were adding each component, as we were trying to avoid breaking the fibers.
  6. As each sample and blank finished incubating, we:
    1. Spun down the AuNP fiber sample and chymotrypsin blank at 12,000 RPM for 1 minute
    2. Combined, in a 600µL Eppindorf tube, the following:
      1. 20uL of the blank or sample
      2. 140uL of Assay Buffer
      3. 40uL of Assay Reagent
      4. Measured the fluorescence with an excitation wavelength of 390nm and an emission spectrum from 400 to 648.5nm.

Data

Figure 1: Raw Data: Fluorescence of alpha-Chymotrypsin Blanks as a Function of the Wavelength of Incident Light (nm)

The figure above shows the raw data for the fluorescence of the alpha-chymotrypsin blanks as a function of the wavelength of incident light. The different curves on the graph represent different incubation times, as indicated by the legend.


Figure 2: Raw Data: Fluorescence of AuNP Fiber Samples as a Function of the Wavelength of Incident Light (nm)

The figure above shows the raw data for the fluorescence of the AuNP fiber samples, which were incubated in alpha-chymotrypsin, as a function of the wavelength of incident light. The different curves on the graph represent different incubation times, as indicated by the legend.


Figure 3: Fluorescence Intensity of the alpha-Chymotrypsin Blanks and the AuNP Fiber Samples as a Function of the Incubation Time (min)

The above figure shows the fluorescence intensity of both the alpha-chymotrypsin blanks (in blue) and the AuNP fiber samples (in red) as a function of the amount of time that the solutions were incubated. The fluorescence intensity was determined by doing the following:

  1. I corrected the alpha-chymotrypsin blanks for instrumental noise by subtracting the fluorescence for the last 0.5nm from all of the other fluorescence measurements for each blank
  2. I did the same correction for instrumental noise for the AuNP fiber samples
  3. I integrated the area under the fluorescence curve for each blank starting at 420nm. This gave the fluorescence intensity of each blank.
  4. I integrated the area under the fluorescence curve for each sample starting at 420nm. This gave the fluorescence intensity of each sample.
  5. I graphed both integrations


Figure 4: Fluorescence Intensity of the AuNP+Peptides as a Function of the Incubation Time (min)


The above figure shows the fluorescence intensity of the AuNP+peptides as a function of the amount of time that the solutions were incubated. The fluorescence intensity was determined by subtracting the fluorescence intensity of the alpha-chymotrypsin blanks from that of the AuNP fiber samples. This effectively subtracted out any fluorescence intensity in the fiber samples that would have been from the alpha-chymotrypsin, giving only the fluorescence intensity of any AuNP that might be in solution and the chymotrypsin-digested AuNP fibers (peptides).


Figure 5: Concentration of AuNP+Peptide (mg/mL) as a Function of Incubation Time in alpha-Chymotrypsin (min)

The above figure shows the concentration of the AuNP+peptides as a function of the amount of time that the solutions were incubated. The concentration of the AuNP+peptides was determined from the data in Figure 4: these data points were divided by the slope of the calibration curve from September 30 (figure 4), which was 475965. This converted the fluorescence intensity to the concentration of AuNP+peptides.


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