User:Andrew W Long/Notebook/CHEM-671 Research/2015/09/22

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Bradford Assay

Lysozyme stock solution (25.85mg) in buffer titrated to 7.505 with 1M HCl

[Tris buffer]: (25.85 mg)/( 5mL) => 5.17 mg/mL => 5,170 μg/mL

The stock was then diluted to 50ug/mL using distilled water.

M1*V1=M2*V2 => (5170 μg/mL)*(V1)=(50 μg/mL)*(50 mL) => V1 = 0.580 μL

6 Assay samples were then created from diluted stock


*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


*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


*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


*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


*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


*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


Detailed instructions can be found in Dr. Harting's notebook here. The one modification to the procedure here is that the sample volumes were 3 mL (as opposed to 1mL), and 25mg of lysozyme was added to a total of 5mL of buffer.


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