User:Jacob Esenther/Notebook/Chem 571/2014/09/10

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  1. Explore the use of Bradford Analysis with Lysozyme


  1. Bradford Analysis
    1. Prepared 50 mL of a standard saline solution (0.9 wt-% NaCl).
    2. Prepared 50 mL of a 50 mM Tris (not Tris-HCl) 50 mM NaCl solution.
    3. Prepared a stock solution of BSA that is roughly 5 mg in 5 mL of saline.
    4. Calculated your actual solution concentration.
    5. Using a quartz cuvette, recorded UV-VIS spectra between 200 nm and 800 nm.
    6. Prepared serial dilutions of 1μg/mL, 2μg/mL, 2.5μg/mL, 5μg/mL, 8μg/mL, 10μg/mL, 15μg/mL and 20μg/mL
    7. Added 20μL of the serial dilutions to a 1.5mL centrifuge tube
    8. Added 200μL of the Bio-Rad Protein Assay reagent. Use 1:4 concentrate diluted with water.
    9. Added 780μL of Tris/NaCl buffer to make the final volume 1mL.
    10. Obtained a UV-VIS spectrum.
      • Blanks of Tris/NaCl buffer and 200 μLBradford reagent + 800μL buffer were also run


Bradford Assay with Lysozyme
Bradford Absorbance vs. Concentration
Bradford Absorbance vs. Concentration (outliers removed)
BSA and Lysozyme Stock Solutions for Bradford
Corrected Bradford-BSA Absorbance


No notes to report