User:Rebeca Rodriguez/Notebook/Chem 471/2015/09/23

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Objective

Calibration curve for Bradford Assay + lysozyme

Protocol

Instructions taken from Dr. Hartings.

The basic protocol that we will be using for this procedure can be found here. (*Note: use section 2.3, page 5)

  1. Make a stock solution that is roughly 10mg protein in 5mL of buffer.
  2. Calculate what your actual concentration
  3. Make 6 standard solutions (3mL each) between 1μg/mL and 10μg/mL
    1. Determine the appropriate volume of your stock to use (for the proper final concentration in 3mL) and add that volume to a plastic cuvette.
    2. Add 600μL of the Bio-Rad Protein Assay reagent
    3. Add the correct amount of buffer such that the final volume is 3mL
    4. Close the cuvettes with parafilm and invert them to mix.
    5. Let them sit for 5 minutes
  4. Take a UV-Vis (no less than 1 hour after they were produced).
    1. Use the plastic cuvettes.
  5. Make a blank as well (2400uL buffer and 600uL Assay reagent) and take it's UV spectrum. (400nm-800nm)
  6. After you have finished one set, repeat the process (make new samples and new measurements)
  7. Make a calibration curve.
  8. Determine if you need to redo any data or sample prep.

Sample Preparation

To make the stock solution, 10.1 mg Lysozyme was added to a 5 mL volumetric flask. The volumetric flask was then filled to the line with buffer solution (50 mM Tris 50 mM NaCl pH 7.5). The final stock solution had a concentration of 2020 μg/mL. -- Made by Michael.


The first round of standard solutions were prepared using the volumes of solution found below.

Final Concentration (μg/mL) Volume of Stock solution (μL) Volume of Bradford Reagent (μL) Volume of Buffer (μL)
1.0 1.48 600 2398.52
2.5 3.71 600 2396.29
5.0 7.42 600 2392.58
7.5 11.13 600 2388.87
8.5 12.62 600 2387.38
10 14.85 600 2385.15
Blank 0 600 2400

The second round of standard solutions were prepared using the volumes of solution found below. The solutions described below used 1:4 diluted Bradford reagent resulting in brilliant blue solutions which become darker with increased protein concentration.

Final Concentration (μg/mL) Volume of Stock solution (μL) Volume of Bradford Reagent (μL) Volume of Buffer (μL)
0 (blank) 0 150 2850
1.01 1.50 150 2848.5
2.36 3.50 150 2846.5
5.05 7.50 150 2842.5
7.41 11.0 150 2839
8.75 13.0 150 2837
9.76 14.5 150 2835.5

Results

Data and Analysis taken from [User:Michael S. Bible/Notebook/CHEM-671/690 Lab Notebook/2015/09/23|Michael Bible], he started all of the data before I got there.

Figure 1: The figure above shows the UV-Vis spectra of the Bradford solution mixed with varying concentrations of lysozyme.

Figure 2: The figure above is a calibration curve for the concentration of lysozyme using a Bradford Assay constructed from the spectra in Figure 1. It is clear that something went wrong with this experiment because there should be a linear relationship between concentration and absorbance at λ = 600 nm. The extinction coefficient for Bradford bound to lysozyme at λ = 600 nm, according to this graph, is ε = 0.0231 mL·μg-1·cm-1.

Figure 3: The figure above shows the UV-Vis spectra of the diluted Bradford solution mixed with varying concentrations of lysozyme.

Figure 4: The figure above is a calibration curve for the concentration of lysozyme using a diluted Bradford Assay constructed from the spectra in Figure 3. It is clear that something went wrong with this experiment because there should be a linear relationship between concentration and absorbance at λ = 600 nm. The extinction coefficient for Bradford bound to lysozyme at λ = 600 nm, according to this graph, is ε = 0.0224 mL·μg-1·cm-1.

Notes

It was noted that during the second round of Bradford analysis of lysozyme using the diluted Bradford reagent, particles were observed floating in the higher concentration lysozyme samples. This suggests that the lysozyme crashed out of solution before the UV-Vis spectra were taken. This could explain why a linear relationship was not observed. The calibration curve should be remade. Perhaps the solutions were prepared and left to sit too long prior to analysis with UV-Vis.


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