User:Sydney Marshall/Notebook/Protease Project/2015/09/22

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Objective

Perform a Bradford Assay analysis on Lysozyme using UV-VIS in order to generate a calibration curve.

Bradford Assay

  • Bradford Assay
    • Mass of Lysozyme stock solution = 25.85mg
    • Buffer titrated to pH of 7.505 with 1M HCl
    • [Tris buffer]: (25.85 mg)/( 5mL) => 5.17 mg/mL => 5,170 μg/mL
    • Stock was then diluted to 50ug/mL using distilled H2O.
    • M1*V1=M2*V2 => (5170 μg/mL)*(V1)=(50 μg/mL)*(50 mL) => V1 = 0.580 μL
    • Then 6 Bradford assay samples were created from diluted stock. Calculations are below.
  • 1μg/mL
    • 600 μL of Bio-Rad Protein Assay reagent
    • M1*V1=M2*V2 => (5170 μg/mL)*(V1)=(1 μg/mL)*(3 mL) => 0.06 mL => 60 μL diluted Lysozyme stock
    • 3mL total - 0.06mL stock - 0.6 mL reagent = 2.34 mL water needed
  • 3μg/mL
    • 600 μL of Bio-Rad Protein Assay reagent
    • M1*V1=M2*V2 => (5170 μg/mL)*(V1)=(3 μg/mL)*(3 mL) => 0.18 mL => 180 μL diluted Lysozyme stock
    • 3mL total - 0.180 mL stock - 0.6 mL reagent = 2.22 mL water needed
  • 4μg/mL
    • 600 μL of Bio-Rad Protein Assay reagent
    • M1*V1=M2*V2 => (5170 μg/mL)*(V1)=(4 μg/mL)*(3 mL) => 0.24 mL => 240 μL diluted Lysozyme stock
    • 3mL total - 0.24mL stock - 0.6 mL reagent = 2.16 mL water needed
  • 5μg/mL
    • 600 μL of Bio-Rad Protein Assay reagent
    • M1*V1=M2*V2 => (5170 μg/mL)*(V1)=(5 μg/mL)*(3 mL) => 0.3 mL => 300 μL diluted Lysozyme stock
    • 3mL total - 0.3mL stock - 0.6 mL reagent = 2.1 mL water needed
  • 6μg/mL
    • 600 μL of Bio-Rad Protein Assay reagent
    • M1*V1=M2*V2 => (5170 μg/mL)*(V1)=(6 μg/mL)*(3 mL) => 0.36 mL => 360 μL diluted Lysozyme stock
    • 3mL total - 0.36mL stock - 0.6 mL reagent = 2.04 mL water needed
  • 10μg/mL
    • 600 μL of Bio-Rad Protein Assay reagent
    • M1*V1=M2*V2 => (5170 μg/mL)*(V1)=(10 μg/mL)*(3 mL) => 0.6 mL => 600 μL diluted Lysozyme stock
    • 3mL total - 0.6mL stock - 0.6 mL reagent = 1.8 mL water needed