User:Noah Benjamin/Notebook/Experimental Biological Chem/2011/09/14

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Objective

Description

41 µL of Bovine Serum Albumin EMD Biomedicals Inc.; Fraction V) and 595.9 µL of distilled water were combined to create 6 mL of a 9.97 µg/mL stock solution. 6 disposable cuvettes were filled with 800, 600, 400, 300, 200, and 100 µL of the BSA stock solution respectively. Each cuvette was then filled with 200 µL of Bradford Reagent (BioRad Laboraties Inc.) and enough distilled water to produce a final solution volume of 1 mL. 10 µL of Maltose binding protein (generously provided by Dr. Hardings) was combined with 990 µL of distilled water as a stock solution. 200 µL of this protein stock solution was combined in a cuvette with 200 µL of Bradford Reagent and 600 µL of distilled water. Blank controls were then prepared by preparing two cuvettes of 200 µL Bradford Reagent and 200 µL of the protein stock solution respectively, and adding 800 µL of distilled water to each. UV-Visible spectra analyses were then taken of each cuvette using a UV-2550 Spectrophotometer (Shimadzu). The spectra were analyzed and calibration curves were prepared.

Data

Image:AbsvsWavelength.png

Image:Protein"Blank"Absorbance.png Graph of the "blank" protein in water Absorbance vs Wavelength.

Image:AdjustedCalibrationCurve.png This calibration curve was created from the standard solutions. However, the last point was removed for accuracy and the intercept was adjusted to 0.

Image:Protein"Blank"MolarAbsorbtivity.png Molar absorbtivity of the protein vs. wavelength derived from Protein "blank" Absorbance and the concentration (0.5321M)from the calibration curve.


What I need to see is a spectrum that shows just MBP in just water/buffer. Also, the concentration that you calculated, is that for the concentrated protein sample or the sample that you measured? Matt Hartings 21:19, 20 September 2011 (EDT)

Notes

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