User:Megan L. Channell/Notebook/Horseradish/2013/09/10

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Horseradish Peroxidase UV-Vis and Gold Solution

Objective

Determine Horseradish Peroxidase (HRP) concentration through a calibration curve. This is done through the Bradford Assay.

Protocol

  1. Make a stock solution with 10.4 mg HRP in 2 mL of Tris buffer.
  2. Actual caluclulated concnetration 5200μg/mL
  3. Standards were made, 1.9 μg/mL, 2.2 μg/mL, 2.7 μg/mL, 3 μg/mL, 3.3 μg/mL, 7 μg/mL
    1. Deteremine appropriate volume of stock. (Ex. Standard 1.9, 3.65 μL added)
    2. Add 200 μL of Bio-Rad Protien Assay reagent
    3. Add buffer to reach a total volume of 1 mL
    4. Shake the eppendorf tube
  4. Take a UV-Vis with plastic cuvettes
  5. Make two blanks with 800μL buffer and 200 μL Assay reagent
  6. Repeat the entire process for a total of two trials
  • Tris Buffer iwas 50.02mM Tris and 50.52mM NaCl

Gold AA/ICPMS Standards

  1. 25 μg/mL
  2. 20 μg/mL
  3. 15 μg/mL
  4. 10 μg/mL
  5. 5 μg/mL
  • A 10 mL volumetric flask was used to make these standards
  • Note water is used to make solutions
  • Stock Solution 1000±10μg/mL

Data

  • UV-VIs HRP for Trial 1 and 2

Image:2013 cmj trial1,2.png

  • Calibration curve of HRP for Trial 1 and 2

Image:2013 cmj calitrial1,2.png

Note

Trial 1 and 2 were done with a cuvette that had a path length of .412 cm. Trial 1 and 2 were not accurate when looking at the calibration curve. A new set of standards will be made for tomorrow.


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