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

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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.
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.


==Description==
==Protocol==
# Add experimental record here. Include what, how, and why...
 
*Preparation of the stock solution (4ml - 10µg/ml)
#Add 0.027ml of BSA (1.46mg/ml) to 3.973ml of water
#Vortex the solution
 
*Preparation of the 6 standard solutions (1ml)
 
[[Image:14sept - Tableau SSolution.png]]
 
*Dilution to 1/1000 of the MPB solution
# Add 0.1ml of pure MPB to 0.9ml of water (dilution to 1/10 of pure solution)
# Add 0.1ml of 1/10 MPB diluted solution to 0.9ml of water (dilution to 1/100 of pure solution)
# Add 0.1ml of 1/100 MPB diluted solution to 0.9ml of water (dilution to 1/1000 of pure solution)
 
*Bradford Assay Technique
# Add 1ml of standard solution (1,2,3,4,5 and 6) to 200µl of Bradford reagent and measure absorbance at 595nm
# Add 1ml of MPB diluted solution (1/100 and 1/1000) to 200µl of Bradford reagent and measure absorbance at 595nm
# Add 1ml of water to 200µl of Bradford reagent and measure absorbance at 595nm (blank)
# Measure absorbance at 595nm of only MPB diluted solution (1/100 and 1/1000)


==Data==
==Data==

Revision as of 04:25, 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)

Data

  • Add data and results here...

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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