User:Matthew R Skorski/Notebook/471 - Exp BioChem/2015/09/22

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Objective

The goal for the day was to perform a Bradford Assay analysis on Lysozyme using UV-VIS and generate a calibration curve.

Description

The instructions for the day can be found here with additional instructions on the Bradford Assay here. A change from the original instructions is that the samples created would be 3 mL, not just 1 mL. Also, 25 mg of lysozyme was added to a total of 5 mL of buffer.

  1. Making Lysozyme Stock Solution
    1. Lysozyme Solution
      1. Actual mass was 25.85 g
      2. After adding Lysozyme to 5 mL of 100mM Tris 50 mM CaCl2 pH 7.4 buffer concentration was (25.85 mg)/( 5mL) => 5.17 mg/mL => 5,170 μg/mL
      3. Diluted stock down further to 50 μg/mL with distilled water to make mixing possible
        1. M1*V1=M2*V2 => (5170 μg/mL)*(V1)=(50 μg/mL)*(50 mL) => V1 = 0.580 μL
      4. Created 6 Bradford Assay samples from diluted stock
        1. 1μg/mL
          1. 600 μL of Bio-Rad Protein Assay reagent
          2. M1*V1=M2*V2 => (5170 μg/mL)*(V1)=(1 μg/mL)*(3 mL) => 0.06 mL => 60 μL diluted Lysozyme stock
          3. 3mL total - 0.06mL stock - 0.6 mL reagent = 2.34 mL water
        2. 3μg/mL
          1. 600 μL of Bio-Rad Protein Assay reagent
          2. M1*V1=M2*V2 => (5170 μg/mL)*(V1)=(3 μg/mL)*(3 mL) => 0.18 mL => 180 μL diluted Lysozyme stock
          3. 3mL total - 0.180 mL stock - 0.6 mL reagent = 2.22 mL water
        3. 4μg/mL
          1. 600 μL of Bio-Rad Protein Assay reagent
          2. M1*V1=M2*V2 => (5170 μg/mL)*(V1)=(4 μg/mL)*(3 mL) => 0.24 mL => 240 μL diluted Lysozyme stock
          3. 3mL total - 0.24mL stock - 0.6 mL reagent = 2.16 mL water
        4. 5μg/mL
          1. 600 μL of Bio-Rad Protein Assay reagent
          2. M1*V1=M2*V2 => (5170 μg/mL)*(V1)=(5 μg/mL)*(3 mL) => 0.3 mL => 300 μL diluted Lysozyme stock
          3. 3mL total - 0.3mL stock - 0.6 mL reagent = 2.1 mL water
        5. 6μg/mL
          1. 600 μL of Bio-Rad Protein Assay reagent
          2. M1*V1=M2*V2 => (5170 μg/mL)*(V1)=(6 μg/mL)*(3 mL) => 0.36 mL => 360 μL diluted Lysozyme stock
          3. 3mL total - 0.36mL stock - 0.6 mL reagent = 2.04 mL water
        6. 10μg/mL
          1. 600 μL of Bio-Rad Protein Assay reagent
          2. M1*V1=M2*V2 => (5170 μg/mL)*(V1)=(10 μg/mL)*(3 mL) => 0.6 mL => 600 μL diluted Lysozyme stock
          3. 3mL total - 0.6mL stock - 0.6 mL reagent = 1.8 mL water
      5. Ran samples through UV-VIS along with a two blanks just containing 2400uL buffer and 600uL reagent

Results

Image:2015 9 22 Lysozyme Absorbance Raw.png This graph shows the absorbance of the samples as function of the wavelength of incident light. The raw data for the six Lysozyme samples and blank are shown.

Image:2015 9 22 Lysozyme Absorbance Corrected.png This graph shows the absorbance of the samples as function of the wavelength of incident light when corrected. The six samples were first corrected for by subtracting the absorbance of the blank from every wavelength of the samples. Then, the isosbestic point at 535 nm from each samples was subtracted from all the wavelengths of that sample.

Image:2015 9 22 Lysozyme Calibration Curve.png

This graph shows the calibration curve for the absorbance of the samples as function of the concentration of Lysozyme in μg/mL. Each peak shows the corrected absorbance of the sample at 600 nm. It is noticed that 10 μg/mL point deviates from the general trend, affecting the R2 value.

Image:2015 9 22 Lysozyme Calibration Curve Remove 10.png

This graphs shows the same information as the graph above except for the remove of the 10 μg/mL sample. The R2 value increases significantly.




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