User:Michaela Harper/Notebook/Biological Chemistry Lab/2011/09/13

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Objective

Perform a Bradford Assay to determine the concentration of an unknown concentration of Maltose Binding Protein (MBP).

Description

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

This graph shows the linear relationship between concentration and absorbance for a BSA standard solution. The stock solution was 1.46 mg/mL. This was diluted by 100 to get a 0.0146 mg/mL solution. This was used to make standard solutions of 1, 2, 4, 6, 8, and 10 ug/mL. The absorbance at 595 nm is plotted against these concentrations.

Image:BSA_linearity.png

The R2 value for the linear regression is 0.9958. The equation for the line is shown on the graph. This equation was used to determine the concentration of the unknown MBP. We used the corrected absorbance at 595 nm as the y value and solved for x. We also incorporated the fact that we diluted the original stock solution by 1000.

This graph shows the calculated molar absorptivity vs. wavelength for MBP without Bradford reagent. The molar absorptivity was calculated using the equation, ε = A / bc where ε is molar absorptivity, A is absorbance, b is the pathlength and c is concentration. The pathlength of the cell is a 1 cm cuvette. The concentration was calculated as the diluted concentration of the standard we used, determined by the information from the BSA graph and line equation.

Image:Molar_absorptivity.png

Image:280nm_zoom_MBP.png

The protein in this sample is waaaaay over-concentrated. For protein spectra, we need a graph that shows features at 280nm. Matt Hartings 21:37, 20 September 2011 (EDT)

Notes

Calculations:

To determine volume of diluted standard (0.0146 mg/mL) needed to make 1 mL samples of desired concentrations (1, 2, 4, 6, 8, and 10 ug/mL):

For 10 ug/mL

M1V1 = M2V2

(0.0146 mg/mL) V1 = (0.01 mg/mL) (1 mL)

V1 = 0.685 mL

Repeated for 1, 2, 4, 6 and 8 ug/mL.


These volumes were pipetted out into cuvettes and water was used to create a total of 1 mL solutions for each. 200 uL were removed from each and replaced with Bradford reagent.


To determine the concentration of MBP:

Absorbance at 595 nm = 0.077

y = 0.0487x + 0.0247

0.077 = 0.0487x + 0.0247

x = 1.074 ug/mL --> diluted concentration from stock solution

1.074 x 1000 = 1.074 mg/mL --> original stock solution concentration



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