User:Douglas M. Fox/Notebook/AU CHEM-571 F2011 Lab Support/2014/09/08

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Test of Bradford Assay

  1. Prepared BSA stock solution
    • 10.19 mg added to 10 mL vol flask (standard DI water used)
    • 1.02 g/L = 15.4 μM (66,430 Da)
  2. Prepared 250 mL 50 mM Tris 50 mM NaCl
    • 2.49605 g Tris = 82.4 mM (121.14 g/mol) --> note error: should have used 1.5 g!!
    • 0.72526 g NaCl = 49.6 mM (58.44 g/mol)
  3. Used 11/2011 Bradford Assay reagent in Rm 207 fridge (undiluted!!)
  4. Used different pipetter for each reagent
    • 0.5 - 10 μL for protein stock (set at 1, 2, 4, 6, 8, or 10 μL)
    • 20 - 200 μL for Bradford reagent (set at 100 μL)
    • 200 - 1000 μL for buffer (set at 800, 799, 798, 796, 794, 792, or 790 μL)
  5. Vortexed 10 s and let sit for 3 - 5 min
  6. Transferred to PS cuvette and measure Vis spectrum 400 nm - 800 nm
    • 6 μg/mL appeared to be outlier; remeasured and used average of two measurements
    • tested previously prepared BSA & Lysozyme solutions, too
  7. Measured UV-VIS of pure protein solutions in quartz cuvettes, from 200 nm - 800 nm

Analysis of Results

Used UV-Vis of protein stock solution to determine protein purity

UV-Vis BSA Stock.png


From plot

  • A278 nm = 0.651
  • ε = 43,824 M-1cm-1
  • [BSA] = 1.49 x 10-5 = 0.987 g/L
  •  %-purity = 96.8%


Difference Spectra of Bradford - BSA solutions

Bradford Difference Spectra.png


Bradford Assay Calibration Curve

Bradford Calibration Curve.png


μg/mL = (A - 0.071)/0.0408

Conclusions

  • Multiple measurements are likely; significant error for 6 μL
  • Removing the origin improved the calibration curve
  • Small volumes (0.5 - 10 μL) are difficult to measure consistently
  • Bradford analysis still appears accurate


Protein solutions are unstable and should be freshly prepared each week!!