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
- Make a stock solution with 10.4 mg HRP in 2 mL of Tris buffer.
- Actual caluclulated concnetration 5200μg/mL
- 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
- Deteremine appropriate volume of stock. (Ex. Standard 1.9, 3.65 μL added)
- Add 200 μL of Bio-Rad Protien Assay reagent
- Add buffer to reach a total volume of 1 mL
- Shake the eppendorf tube
- Take a UV-Vis with plastic cuvettes
- Make two blanks with 800μL buffer and 200 μL Assay reagent
- Repeat the entire process for a total of two trials
- Tris Buffer iwas 50.02mM Tris and 50.52mM NaCl
Gold AA/ICPMS Standards
- 25 μg/mL
- 20 μg/mL
- 15 μg/mL
- 10 μg/mL
- 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
- Calibration curve of HRP for Trial 1 and 2
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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