User:Elizabeth Ghias/Notebook/Experimental Biological Chemistry/2011/09/14

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Objective

To determine the concentration of an unknown protein by performing a Bradford assay.

Description

  1. A 3 mL stock solution of 14.6 ug/mL BSA in water was made from 1.46 mg/mL concentrated BSA.
  2. 1 mL standard solutions with the concentrations 10,8,6,4,2,1,0 ug/mL were made from the 3 mL stock solution of 14.6 ug/mL BSA
  3. Each solution was mixed with 200 uL of the Bradford reagent for protein assay and placed in a plastic cuvette.
  4. An unknown MBP protein sample was prepared by mixing 1 uL of concentrated MBP with 800 uL of water and 200 uL of the Bradford reagent.
  5. All of the samples were analyzed through UV-vis.


Data

Image:Sept_14_Table_of_Concentrations.png

Image:Absorbance_vs._Concentration_September_14.png

Grubb's outlier test was performed to determine if the last data point in the plot was an outlier. The calculations determined that the point was not an outlier. The intercept of the linear regression for the plot has a value of 0.1782. Ideally, the intercept should be at the origin and have a value of 0. The linear regression was used to determine the concentration of the unknown MBP sample by using the corrected absorbance of the unknown sample at 550, which was equal to 0.113. The concentration of the 1000X diluted unknown was calculated to be 0.960 mg/mL. The concentration of the undiluted protein sample was equal to 0.960 g/mL or 1.4 x 10 M. The molar absorptivity of the MBP sample in water was graphed against the wavelength.

Image:October_14_Molar_Absorptivity_of_MBP_vs._Wavelength.png


Notes

This area is for any observations or conclusions that you would like to note.


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