User:Floriane Briere/Notebook/CHEM-496/2011/09/14: Difference between revisions

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==Results==
==Results==


[[* Curve of the concentration (nM) as a function of the Absorbance at 595nm]]
'''* Curve of the concentration (nM) as a function of the Absorbance at 595nm'''


[[Image:14sept - Abs(595=f(concentration).png]]
[[Image:14sept - Abs(595=f(concentration).png]]
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So, the pure MPB solution has a concentration of 31.2nM (0.0312*1000 = 31.2nM).
So, the pure MPB solution has a concentration of 31.2nM (0.0312*1000 = 31.2nM).


* Curve of the Absorbance as a function of the Wavelength (nm)
'''* Curve of the Absorbance as a function of the Wavelength (nm)'''


[[Image:14sept - Absorbance = f(wavelength) v2.png]]
[[Image:14sept - Absorbance = f(wavelength) v2.png]]
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This curve is made with the 1/1000 diluted MPB solution measures.  
This curve is made with the 1/1000 diluted MPB solution measures.  


* Curve of the molar absorptivity (ε) as a function of the Wavelength (nm)
'''* Curve of the molar absorptivity (ε) as a function of the Wavelength (nm)'''


According to Beer-Lambert law, Absorbance = molar absorptivity (L.mol^-1.cm^-1) * concentration (M) * length of the cuve (cm)
According to Beer-Lambert law, Absorbance = molar absorptivity (L.mol^-1.cm^-1) * concentration (M) * length of the cuve (cm)

Revision as of 06:55, 20 September 2011

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Objective

Today's experiment is to test the Bradford Assay technique which is going to allow us to determine the unknown proteins’ concentration of a solution. We are going to use 6 standards solutions of BSA whose concentrations are known. Then, thanks to a spectrophotometer, we’ll be able to determine the concentration of the MBP solution.

Protocol

  • Preparation of the stock solution (4ml - 10µg/ml)
  1. Add 0.027ml of BSA (1.46mg/ml) to 3.973ml of water
  2. Vortex the solution
  • Preparation of the 6 standard solutions (1ml)

  • Dilution to 1/1000 of the MPB solution
  1. Add 0.1ml of pure MPB to 0.9ml of water (dilution to 1/10 of pure solution)
  2. Add 0.1ml of 1/10 MPB diluted solution to 0.9ml of water (dilution to 1/100 of pure solution)
  3. Add 0.1ml of 1/100 MPB diluted solution to 0.9ml of water (dilution to 1/1000 of pure solution)
  • Bradford Assay Technique
  1. Add 1ml of standard solution (1,2,3,4,5 and 6) to 200µl of Bradford reagent and measure absorbance at 595nm
  2. Add 1ml of MPB diluted solution (1/100 and 1/1000) to 200µl of Bradford reagent and measure absorbance at 595nm
  3. Add 1ml of water to 200µl of Bradford reagent and measure absorbance at 595nm (blank)
  4. Measure absorbance at 595nm of only MPB diluted solution (1/100 and 1/1000)

Results

* Curve of the concentration (nM) as a function of the Absorbance at 595nm

Red lines represent the Absorbance at 595nm of the 1/1000 diluted MPB solution. This curve allow us to determine the concentration of the 1/1000 diluted MPB solution. To draw this curve, we add to take into account that the Bradford Assay was made with 5/6 diluted solutions (because we added 1ml of solution to 200µl of Bradford reagent).

According to this curve, the 1/1000 diluted MPB solution has a concentration of 0.0312nM (0.026*(6/5) = 0.0312nM). So, the pure MPB solution has a concentration of 31.2nM (0.0312*1000 = 31.2nM).

* Curve of the Absorbance as a function of the Wavelength (nm)

This curve is made with the 1/1000 diluted MPB solution measures.

* Curve of the molar absorptivity (ε) as a function of the Wavelength (nm)

According to Beer-Lambert law, Absorbance = molar absorptivity (L.mol^-1.cm^-1) * concentration (M) * length of the cuve (cm)

Notes

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


Use categories like tags. Change the "Course" category to the one corresponding to your course. The "Miscellaneous" tag can be used for particular experiments, as instructed by your professor. Please be sure to change or delete this tag as required so that the categories remain well organized.



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