User:Rebeca Rodriguez/Notebook/Chem 471/2015/09/30

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Bradford Analysis of Protease Degradation

The following protocol was taken from Dr. Hartings's lab notebook


All samples will be incubated in eppendorf tubes, which will be placed in the 35C water bath on the shaker. Samples should all contain the same concentration of materials.

Samples will be prepared for measurements at 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hr, 1.5 hr, 2 hr.

  1. Protease Prep
    1. Place the 1mL of Tris/CaCl2 buffer in the pre-measured protease sample (0.95 mg Proteinase K) and record the concentration (3.28·10-5 M)
  2. Sample Prep
    1. To a dried fiber sample tube (from previous fiber syntheses in the eppendorf tubes) add the appropriate amounts of buffer and protease (30.5μL Proteinase K, 969.5 μL Buffer)
      1. The total volume should be 1mL
      2. The final protease concentration should be 1uM
      3. Vortex the sample to disperse the fibers
  3. Blank Prep (one blank to match each sample)
    1. To a clean eppendorf tube add the appropriate amounts of buffer and protease (30.5μL Proteinase K, 969.5 μL Buffer)
      1. The total volume should be 1mL
      2. The final protease concentration should be 1uM
  4. Incubate Sample and Blanks
    1. Place the tubes in the shaker with the 37C water bath
  5. Measurement
    1. Remove the tubes (the sample and a corresponding blank) from the water bath at the appropriate time
    2. For the case of a sample with fibers, centrifuge the sample for 1 min to pull any fibers to the bottom of the tube
    3. To a plastic cuvette add
      1. 600 uL of pre-mixed Bradford dilution (1:3 Bradford Reagent:Buffer)
      2. 750 uL of sample
      3. 1650 uL of buffer
    4. Record the UV-Vis spectrum from 400-800 nm
  6. Using the Bradford standardization curve and data from the 0 fiber sample, determine the concentration of released protein in your solution.

Note

Due to an error in buffer labeling, a phosphate buffer was used to create the stock protease solution. The stock protease solution for today was created by adding 1 mL of phosphate buffer to 95 mg of Proteinase K. This stock solution had a concentration of 3.28·10-5 M. Thus each sample solution was made by adding 30.5 μL of proteinase K solution and 969.5 μL of Tris/CaCl2. Because the Proteinase K solution was made from phosphate buffer, when it was added to the Tris/CaCl2 solution in each sample and blank solution, calcium phosphate crashed out of the solution. This could have interfered with the results obtained.

Discussion

Spectra above shows Proteinase K-Degraded AuNP Fiber samples that have been allowed to chop up the fibers for different amounts of time. The spectra were created by subtracting each spectrum's respective blank spectrum and by adjusting all points so that the graphs had zero absorbance at λ = 535 nm.


The figure above shows the absorbance values of each of the degraded fiber samples at λ = 600 nm. Negative absorbance values are not covered by the Bradford Calibration curve created on 9/23/15. The fact that all absorbance values are negative suggests that there was no protein left in the solution after degradation. It is also possible that centrifuging the solutions after incubation pulled all of the protein in solution down to the bottom of the tubes. If this was the case, it could explain why no protein was found in the solution after incubation.


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