User:Nicole Bonan/Notebook/Chem 571 Lab Notebook/2015/10/06

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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

  1. 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.
  2. Next, we prepared AuNP fiber samples for use in our experiment.
    1. Dr Hartings had synthesized the AuNP fiber samples on October 2nd.
    2. We had seven AuNP samples total, one for each incubation time that we were interested in:
      1. 10 min
      2. 15 min
      3. 30 min
      4. 45 min
      5. 1 hour
      6. 1.5 hours
      7. 2 hours
    3. 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.
    4. We pipetted off as much supernatant as we could.
  3. 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).
    1. 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
    2. 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
  4. 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.

Sample Prep To a fiber sample tube (that has been centrifuged and had the supernatant removed) add the appropriate amounts of buffer and protease The total volume should be 1mL The final protease concentration should be 1uM Vortex the sample to disperse the fibers Blank Prep (you will have one blank to match each sample) To a clean eppendorf tube add the appropriate amounts of buffer and protease The total volume should be 1mL The final protease concentration should be 1uM Incubate Sample and Blanks Place the tubes (for both the Blanks and Samples) in the shaker with the 37C water bath Measurement Remove the tubes (the sample and a corresponding blank) from the water bath at the appropriate time For the case of a sample with fibers, centrifuge the sample for 1 min to pull any fibers to the bottom of the tube Blank measurement From the blank measurement, take 20uL of blank and place in a 600uL eppendorf tube Add 140uL of Assay Buffer Add 40uL of Assay Reagent Take Measurement Sample Measurement From the sample, take 20uL of sample and place in a 600uL eppendorf tube Add 140uL of Assay Buffer Add 40uL of Assay Reagent Take Measurement Excitation at 390 nm Emission from 400 to 650 nm

Data


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