User:Douglas M. Fox/Notebook/AU CHEM-571 F2011 Lab Support/2014/09/08: Difference between revisions
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== | ==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 | |||
* A<sub>278 nm</sub> = 0.651 | |||
* ε = 43,824 M<sup>-1</sup>cm<sup>-1</sup> | |||
* [BSA] = 1.49 x 10<sup>-5</sup> = 0.987 g/L | |||
* %-purity = 96.8% | |||
<br> | |||
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!!''' | |||
Revision as of 21:43, 9 September 2014
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Test of Bradford Assay
Analysis of ResultsUsed UV-Vis of protein stock solution to determine protein purity From plot
Bradford Assay Calibration Curve μg/mL = (A - 0.071)/0.0408 ConclusionsMultiple 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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