User:Madeleine Y. Bee/Notebook/CHEM-571 2013F/2013/09/10

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September 10, 2013


Execute the following procedure, performing the Bradford Assay to measure very low protein concentrations. Make standard solutions for Atomic Absorption measurements tomorrow.


Round 1: Bradford Assay with Hemoglobin

  • Measured amount of hemoglobin- 0.010g
  • Concentration of hemoglobin stock solution- 5mg/mL
    • Stock solution made by dissolving hemoglobin in 1.5mL TRIS buffer, which was added to a 2mL volumetric flask. The remaining amount of TRIS buffer necessary to bring the solution up to 2mL was added.
  • Standard solutions:
    • 1ug/mL
    • 2ug/mL
    • 4ug/mL
    • 5ug/mL
    • 8ug/mL
    • 9ug/mL
    • Blank
    • Standard solutions made by combining 200uL Assay reagent, appropriate amount of hemoglobin for desired concentration, and diluting up to 1mL with TRIS buffer. Blank made by combining 200uL Assay reagent with 800uL TRIS buffer.

Round 2: Bradford Assay with Hemoglobin

  • Repeat above procedure. Use same blank for reference.

Standards for Atomic Absorption Measurements (performed tomorrow)

  • Make 5 samples at the following concentrations:
    • 25ug/mL
    • 20ug/mL
    • 15ug/mL
    • 10ug/mL
    • 5ug/mL
    • 5 10mL standard solutions made using 1000Mg/mL gold solution, diluted with water


Absorbance Spectra


Calibration Curves

2013 0919 hemoglobin calibration.PNG