User:Omar Choudary/Notebook/Omar Choudary CHEM-571 AU-2011/2012/2011/09/14

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Objective

  • Perform a Bradford Assay to determine the concentration of an unknown solution of Maltose Binding Protein (MBP)

Description

  • Created 1.2 mL standards using 1, 2, 4, 6, 8, and 10 μg/mL BSA solutions in water with 200 μL Bradford Agent:

Blank - 0.000 μg/mL

Standard 1 (S1) - 0.833 μg/mL

Standard 2 (S2) - 1.667 μg/mL

Standard 3 (S3) - 3.333 μg/mL

Standard 4 (S4) - 5.000 μg/mL

Standard 5 (S5) - 6.667 μg/mL

Standard 6 (S6) - 8.333 μg/mL

  • Created 1 mL samples of unknown MBP by 1/10, 1/100, and 1/1000 serial dilutions, and then added 200 μL bradford reagent.
  • Obtained spectra from 200 - 800 nm of all standards and unknowns using UV-Vis Spectrophotometer (Shimadzu 2550).

Data

  • Spectra of different concentrations of BSA.

OC-9-14-1.jpg


  • Calibration curve of the six standards at 595 nm. The equation was used to calculate the concentration of MBP.

OC-9-14-2.jpg


  • The absorbance of a 1/100 dilution of MBP at 280nm, used to calculate the concentration of MBP using the equation for the BSA standards at 595 nm.

OC-9-14-3.jpg

  • After calculating the concentration of MBP, the molar absorptivity of MBP could be calculated for the entire spectrum.

OC-9-14-4.jpg

Notes

  • Corrected Absorbance of MBP at 280nm = 0.163
  • Using equation for BSA/Bradford --> 0.163 = 0.0315x - 0.0205

x = concentration of MBP x = 5.8253 μg/mL

  • MBP is 1/10 dilution therefore 58.253 μg/mL is concentration of undiluted MBP.

58.253 μg/mL * 1000 mL / 1 L * 1 g / 1000000 μg * 1 mole MBP / 66776 g MBP = 8.723 * 10^-7 M

  • Using beer's law molar absorptivity was calculated for the entire MBP spectrum.

A = εbc

ε = A / 8.723*10^-7

At 280nm ε = 196,033 L / cm * mole

  • MBP dilutions used for calculating absorptivity were prepared again on 9/21, due to major inconsistencies in the data.