User:James C. Schwabacher/Notebook/CHEM-571/2013/09/10

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Objective

Utilize the Bradford Assay to make calibration curves for Horseradish Peroxidase (HRP).

Procedure

  1. Make a stock solution of 10.4 mg of HRP in 10mL of buffer
  2. Actual concentratio: 5200μg/mL
  3. Make 6 standard solutions (1mL each) between 1μg/mL and 10μg/mL
      1. 1.9μg/mL
      2. 2.2μg/mL
      3. 3.0μg/mL
      4. 3.3μg/mL
      5. 7.0μg/mL
    1. Determine the appropriate volume of your stock to use (for the proper final concentration in 1mL) and add that volume to an eppendorf tube
    2. Add 200μL of the Bio-Rad Protein Assay reagent
    3. Add the correct amount of buffer such that the final volume is 1mL
    4. Shake
    5. Let them sit for 5 minutes
  4. Take a UV-Vis (no less than 1 hour after they were produced).
    1. Use the plastic cuvettes.
  5. Make 2 blanks as well (800uL buffer and 200uL Assay reagent) and take it's UV spectrum. (400nm-800nm)
    1. We will be using one of the blanks as a reference for each spectrum that we take. I'll show you where to place this cuvette for each spectrum you collect)
  6. After you have finished one set, repeat the process (make new samples and new measurements)

- Note: This is where coordination is a good thing. Take 1 spectrum at a time. Let other people go.

  1. Make a calibration curve.
  2. Determine if you need to redo any data or sample prep.

Note - Solutions from today containing the stain will go into a waste container in the hood.

We are also going to make Atomic Absorption standards for tomorrow!

Using the gold AA/ICPMS standard solution make 5 new solutions (Note: Use water - NOT BUFFER - to make these solutions)

  1. 25 ug/mL
  2. 20 ug/mL
  3. 15 ug/mL
  4. 10 ug/mL
  5. 5 ug/mL

Procedure Adapted from Prof. Hartings

Data

  1. Horseradish Peroxidase UV-Vis Trials 1 and 2

CHEM571 09.10.13 HRP 1 2 UVVIs.png

  1. Horseradish Peroxidase UV-Vis Trial 3

CHEM571 09.10.13 HRP 3 UVVIs.png

  1. Horseradish Peroxidase Calibration Curve Trials 1 and 2

CHEM571 09.10.13 HRP 1 2 calibration.png

  1. Horseradish Peroxidase Calibration Curve Trial 3

CHEM571 09.10.13 HRP 3 calibration.png

Notes

All data collected above was collected using the shortened path length of .412 cm. The data collected from trials 1 and 2 was not accurate, so a trial 3 was collected. Trial 3 was also not accurate, as shown through the calibration curves. When corrected for a set y-intercept of 0, the r-squared values become negative for both calibrations. Also, the procedure was further amended for trial 3, during which the dilutions were made using 10 ml, and then transferred to the cuvette,in order to allow for larger, more accurate measurements of stock solution. We will attempt a fourth data collection tomorrow.

The gold standard solution was made using a small sample of the stock solution diluted in a 10ml volumetric flask with distilled water.