User:Helen L. Slucher/Notebook/CHEM 571/2013/09/10

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Objective

Make calibration curves using the Bradford Assay.

Procedure

  1. Make a stock solution that is roughly 10mg protein in 2mL of buffer.
  2. Calculate your actual concentration
  3. Make 6 standard solutions (1mL each) between 1μg/mL and 10μg/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. Take a UV-Vis (no less than 1 hour after they were produced).
  5. Make 2 blanks as well (800uL buffer and 200uL Assay reagent) and take it's UV spectrum. (400nm-800nm)
  6. After you have finished one set, repeat the process (make new samples and new measurements)

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

Data (Trial 1)

  • Image:Screen shot 2013-10-12 at 2.49.52 PM.png
  1. Series 1: 5 ug/mL
  2. Series 2: 6 ug/mL
  3. Series 3: 7 ug/mL
  4. Series 4: 8 ug/mL
  5. Series 5: 9 ug/mL
  6. Series 6: 10 ug/mL
  • Image:Screen shot 2013-10-12 at 2.50.56 PM.png

Data (Trial 2)

  • Image:Screen shot 2013-10-12 at 2.50.19 PM.png
  1. Series 1: 5 ug/mL
  2. Series 2: 6 ug/mL
  3. Series 3: 7 ug/mL
  4. Series 4: 8 ug/mL
  5. Series 5: 9 ug/mL
  6. Series 6: 10 ug/mL
  • Image:Screen shot 2013-10-12 at 2.51.18 PM.png

Notes

  • All data was collected using the shortened path length of 0.412 cm.
  • Stock solution: 4950 ug/mL
  • Trial 1: 0.0099 g protein
  • Trial 2: 0.012 g protein


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