User:Matt Hartings/Notebook/AU Biomaterials Design Lab/2011/09/13

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Objective

Learn how to maintain an OpenWetWare Notebook.

Description

Today you'll be performing a Bradford Assay to determine the concentration of an unknown protein.

You will make standard solutions of BSA (1mL for each solution) in water of 10, 8, 6, 4, 2, and 1 μg/mL. Using the MM of BSA (66776 g/mol) convert these concentration values into M.


Standards:
Make 1mL solutions of BSA in water at 10, 8, 6, 4, 2, 1 µg/mL. I make 3mL of a 10µg/mL solution (1:1000 dilution of the BSA that comes with restriction enzymes) and then make each standard from this stock.

Assay:
800µL protein sample (standards or unknown)
200µL Bradford reagent
• Mix by vortexing
• Don’t forget to do a blank (water instead of protein sample)
• Measure absorbance of standards and unknowns at 595nm (use 800µL in plastic cuvettes)
• Unknowns may need to be significantly diluted in water; this assay’s dynamic range is only 0-10µg/mL.
• Take an absorbance of just the unknown protein (no bradford reagent).

Data analysis:
• Create a graph of protein concentration vs. absorbance for the BSA standards.
• Generate a linear regression line from the standards and obtain the line’s equation. R2 value should ideally be above 0.97.
• From the absorbances of the unknowns, solve the standard equation to find the protein concentration.
• Determine the molar absorptivity of the unknown protein.

Data

  • Add data and results here...

Notes

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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