The objective for today is to measure the UV absorbance of lysozyme AuNP fibers over various incubation times with alpha chymotrypsin using a bradford assay.
- 1mL stock solution of alpha-chymotrypsin was made by adding 1mL of 50mM Tris buffer (pH=8) to dry protease for a final concentration of 41.016µM.
- The above concentration is too low to accurately pipette the required volume, thus a 1mL of a 1:200 dilution was prepared in phosphate buffer to increase the necessary volume to 4.88uL from this new diluted stock. This gave us a new diluted protease concentration of .20506uM
- 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= 4.88uL
- Each sample and blank tube for incubation requires a total volume of 1mL, thus 1mLtotal - 4.88uL protease = 995.12uL Tris buffer.
Incubation Tube Preparation (Samples and Blanks)
- Fiber samples synthesized by Dr. Hartings were used for total of 10 (5 sample and 5 blank) and organized via the following alpha chymotrypsin incubation times.
- 30min, 1hr, 1.5hr, 2 hr, 23hr
- 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 Tris 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.
Bradford 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 bradford assay reaction mix was prepared in a new plastic absorbance cuvettes and contained the components and volumes shown below.Diluted bradford reagent consisted of a 1/4 dilution of the original bradford stock available in Dr. Harting's lab (not the bradford reagent from Dr. Saldanha's lab).
- 750uL of the incubated blank or sample
- 1650uL of Tris Buffer
- 600uL of diluted Bradford reagent
- UV Absorbance was then immediately measured from 400-800nm
- Note that a spectra with the same parameters was taken for a sample with the same makeup as shown above, with the replacement of the incubated sample with Tris buffer for a bradford blank.
Figure 1 above shows absorbance data for both blanks and samples separately at 600nm along alpha-chymotrypsin incubation time. The absorbance data was corrected separately for both samples and blanks. First, the absorbance of the Bradford blank (bradford reagent and Tris buffer as specified above) was subtracted from the absorbance of each incubation timed sample and blank for each wavelength. Then, again individually, the isosbestic point was corrected for by subtracting the absorbance value at the isosbestic point of 535nm from each data point for each timed sample or blank respectively. This allowed for the absorbance values of all samples and blanks to be 0 at 535nm.
Figure 2 above shows the absorbance data of the AuNP samples over alpha-chymotrypsin incubation corrected for blanks. Blank correction was performed by subtracting the values of the Bradford blank and isosbestic point corrected blank data from the Bradford blank and isosbestic point corrected sample (AuNP fiber tube) data. The increase in absorbance as incubation time increases suggests that as alpha-chymotrypsin digests AuNP fibers samples, peptides and small fiber particles are released into solution.