The objective for today is to measure the fluorescence of lysozyme AuNP fibers over various incubation times with alpha chymotrypsin. This will allow us to calculate the concentration of AuNP Fibers and Peptides in solution based on a calibration curve.
- 1mL stock solution of alpha-chymotrypsin was made by adding 1mL of 50mM phosphate buffer (pH=8) to dry protease for a final concentration of 38.672µM.
- Through M1V1= M2V2 the correct volume of protease needed for a final incubation tube concentration of 1uM and final tube volume of 1mL was determined
- V1= .25859uL
- The above concentration is too low to accurately pipette the required volume, thus a 1mL of a 1:20 dilution was prepared in phosphate buffer to increase the necessary volume to 5.17uL from this new diluted stock.
- Each sample and blank tube for incubation requires a total volume of 1mL, thus 1mLtotal - 5.17uL protease = 994.8uL phosphate buffer.
Incubation Tube Preparation (Samples and Blanks)
- Fiber samples synthesized by Dr. Hartings were used for total of 12 (6 sample and 6 blank) and organized via the following alpha chymotrypsin incubation times.
- 10min, 15min, 30min, 45min, 1hr, 1.5hr, 2 hr
- First, AuNP fiber samples were centrifuged at 300 rpm for 10 minutes after which as much supernatant was drained as possible.
- Then, using volumes indicated above, alpha chymotrypsin and phosphate buffer were added to each sample and blank tube immediately before incubation start time.
- Sample and blank pairs were incubated at 37˚ C in a water bath for the noted incubation times above.
Fluorescence Assay Preparation
- After incubation of a sample and blank pair, they were centrifuged at 12,000rpm for 1 min.
- Spun down the AuNP fiber sample and chymotrypsin blank at 12,000 RPM for 1 minute
- Then the fluorescence assay reaction mix was prepared in a new 1.5ml eppendorf tube and contained the components and volumes shown below.
- 20uL of the blank or sample
- 140uL of Assay Buffer
- 40uL of Assay Reagent
- Fluorescence was then immediately measured with a excitation wavelength of 390nm with a recorded emission spectra from 400-649.5nm.
Figure 1 above shows end wavelength corrected fluorescence intensity of both the alpha-chymotrypsin incubated fiber samples and blanks. To correct for instrumental noise, the fluorescence intensity for the last .5nm was subtracted from all fluorescence measurements for each specific blank and sample. Then the area under each fluorescence curve (samples and blanks) was integrated starting at 420 nm and ranging to 649.5nm. Then integrated fluorescence intensity values were plotted for samples and blanks separately.
Figure 2 above shows blank corrected integrated fluorescence intensity over incubation time. The integrated fluorescence intensity of the blanks was subtracted from those of the samples to correct for the blanks. Subtracting our blank fluorescence accounted for fluorescence from the protease (alpha-chymotrypsin), thus this graph shows the fluorescence intensity of AuNPs and peptide fragments resulting from the digest of AuNPs.
Figure 3 above shows the calibration curve derived concentration of AuNP fibers in solution in addition to peptide fragments over protease incubation time. Concentrations were derived from inputting blank and instrumental noise corrected integrated fluorescence values into the calibration curve derived equation for lysozyme from User:Benjamin Friedel/Notebook/CHEM 471/2015/09/29. The equation was y=475965x with y being the fluorescence intensity and x being the concentration. By inputting measured, corrected fluorescence intensities, concentrations were calculated for each incubation time point.