Test of Bradford Assay
- 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)
- 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)
- Used 11/2011 Bradford Assay reagent in Rm 207 fridge (undiluted!!)
- 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)
- Vortexed 10 s and let sit for 3 - 5 min
- 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
- 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
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 Assay Calibration Curve
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!!
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