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

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Objective

  1. Explore the use of Bradford Analysis with Lysozyme

Description

  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


Data

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

Notes

No notes to report




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