Objective
The purpose of today's lab work was to use a Fluorescence Assay in order to determine the rate at which alpha-chymotrypsin digests AuNP fiber samples.
Protocol
- First, we made a 1mL stock solution of our protease, alpha-chymotrypsin, by adding 1mL of phosphate buffer to one of the stock masses of alpha-chymotrypsin that we weight out earlier in the semester. The final molarity of this sample was 47.266µM.
- Next, we prepared AuNP fiber samples for use in our experiment.
- Dr Hartings had synthesized the AuNP fiber samples on October 2nd.
- We had seven AuNP samples total, one for each incubation time that we were interested in:
- 10 min
- 15 min
- 30 min
- 45 min
- 1 hour
- 1.5 hours
- 2 hours
- We spun all of our samples down at 300 RPM for 10 minutes. Our goal was to get the fibers to collect in the bottom of the tube without crushing the fibers together into an unreactive mass.
- We pipetted off as much supernatant as we could.
- After prepping the AuNP fiber samples, we calculated how much alpha-chymotrypsin stock and phosphate buffer we would need to add to each sample and blank. (Our goal was to make one AuNP fiber sample and one alpha-chymotrypsin blank for each of the incubation times listed in step 2.2. All of these solutions would have a concentration of alpha-chymotrypsin of 1µM. All solutions would be brought up to a total volume of 1mL using phosphate buffer).
- To determine how much of our stock of alpha-chymotrypsin we needed:
Let M1=concentration of alpha-chymotrypsin stock=47.266µM V1=volume of alpha-chymotrypsin stock we need to add to the AuNP fiber sample M2=final concentration of alpha-chymotrypsin in the AuNP fiber sample=1µM V2=final volume of alpha-chymotrypsin sample=1mL M1V1=M2V2 V1=(M2V2)/M1 V1=[(1µM)(1mL)]/(47.266µM) V1=0.212mL=21.2µL
- We then subtracted the volume of alpha-chymotrypsin stock we needed to use (21.2µL) from the total volume of the solution (1mL, or 1000µL) in order to determine the volume of phosphate buffer to add:
1000µL-21.2µL=978.8µL of phosphate buffer
- We added the calculated volumes to the samples and blanks. We vortexed all of the AuNP fiber samples (except for the 10min sample) and put all of the samples and blanks into the 37 degree Celsius water bath, except for the 10 minute sample and blank, for their respective incubation times.
- As each sample and blank finished incubating, we:
- Spun down the AuNP fiber sample (but not the blank) at 12,000 RPM for 1 minute
- Combined, in a 600µL Eppindorf tube, to following:
- 20uL of the blank or sample
- 140uL of Bradford Assay Buffer
- 40uL of Bradford Assay Reagent
- Measured the fluorescence with an excitation wavelength of 390nm and an emission spectrum from 400 to 600nm
- We then repeated steps 4-5 for the 10 min sample and blank.
Data
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