Objective
Protocol
Lysozyme Protease Degradation Fluorescence Analysis
On 09/29/15 all of the lysozyme standards were analyzed, today the blank standard were analyzed using the same procedure.
Bradford Analysis of Protease Degradation
- First, a stock solution of our group's protease, alpha-chymotrypsin, was made in 1mL Tris/CaCl2 buffer (pH=8). The concentration of the protease was 40.625µM.
- Second, 7 samples of AuNP fibers (that Dr. Hartings synthesized earlier) were prepared for analysis using the following procedure
- centrifuge samples at 1500rpm for 1 min
- pipette supernatant off of samples (water)
- Samples were then incubated with protease for specific time points, after which Bradford analysis was conducted on them to monitor digestion with UV-Vis. The time points of incubation at 37˚C are as follows.
- 10 min
- 15 min
- 30 min
- 45 min
- 1 hour
- 1.5 hours
- 2 hours
Calculating Protease Volume in Reaction Mix
- To determine what volume of our 40.625uM protease stock solution to add to the reaction mix with the fibers to be incubated the equation M1V1=M2V2 was used with M1= molarity of protease stock, V1= volume of protease stock added to reaction mix, M2= molarity of protease in reaction mix, V2= volume of reaction mix
- The target protease concentration in the reaction mixes was 1uM and the target volume was 1mL, thus the volume of protease stock needed to be added to each reaction mix was 24.6ul.
Calculating Buffer Volume in Reaction Mix
- To determine what volume of Tris-CaCl2 buffer to add to the sample to bring the volume up to 1mL, the volume of protease to be added was subtracted from 1mL as it is assumed that most liquid was removed from the fiber samples after they were centrifuged.
- 1000ul total-24.6ul protease = 975.4ul buffer
- This volume of buffer was added to the spun down and supernatant-removed lysozyme AuNP sample tubes. Protease was not added until a moment before incubation commenced for an individual sample (10min incubation vs. 2 hr).
Blank Preparation
- Blanks were prepared to account for the absorbance of protease alone and protease self digestion
- For each timed sample a blank was prepared and treated in the same way.
- Blanks samples consisted of the same volume of protease and buffer as in the Lysozyme samples, however no lysozyme was added. This maintains the same protease concentration. Again, protease was not added until moments before incubation commenced and blanks were incubated for the same time and in the same conditions (rocking at 37˚C) as their respective lysozyme sample equivalents.
Bradford Analysis
- While we were waiting for the solutions to incubate, we pipetted 600µL of pre-mixed Bradford dilution into each of 14 cuvettes. These cuvettes would be the ones from which we took our UV-Vis measurements.
- When the 15 minute sample and blank were finished incubating, we prepared them for measurement:
- We removed them from the water bath and spun the sample (but not the blank) down at 300 RPM for 1 minute
- We pipetted 1650µL of buffer into each of the cuvettes that would be used for the 15 min sample and the 15 min blank
- We pipetted 750µL of the 15 min sample into the 15 min cuvette. We did the same for the 15 min blank.
- Next, we recorded the UV-Vis spectrum for the two cuvettes from 400-800nm.
- After that, we repeated steps 8-9 for the 30 minute, 45 minute, and 1 hour samples and blanks.
- We then repeated step 5-6 for the 10min sample and blank
- We repeated steps 8-9 for the 1.5 hour sample and blank
- We repeated steps 8-9 for the 10 min sample and blank
- We repeated steps 8-9 for the 2 hour sample and blank
Data
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