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

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Experimental Biological Chemistry: Fall 2013 Main project page
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September 10, 2013

Objective

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

Procedure

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

Data

Absorbance Spectra

700pix

Calibration Curves

Image:2013_0919_hemoglobin_calibration.PNG

Notes

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