LBYBI37 Bradford Assay

• Bradford Assay

  
  

  Colorimetric protein quantitation assay
• Dye used in Bradford Assay

  
  

  Coomassie Brilliant Blue G-250 (CBB G-250)
• pH < 1: positive charge, red/brown color

  1 < pH < 2: no net charge, green color

  pH > 2: negative charge, blue color
• Binding of dye to the protein stabilizes the anionic, blue form of the dye
• Upon initial contact, CBB G-250 donates electrons to the protein, causing a change in the folding of the protein that exposes the protein's hydrophobic regions

  Change in protein conformation allows the dye to further bind to the protein through hydrophobic and Van der Waals interactions
• Bradford reagent is prepared with methanol/ethanol and glacial acetic acid
• UV-Visible Spectrophotometer

  • measures absorbance of transmittance of light in the uv and visible range
  • at 595 nm, protein-bound dye absorbs orange light strongly, protein solution appears blue
  • more dye molecules bound to proteins, more orange light absorbed, more bluish protein solution
• Beer-Lambert Law

  • absorbance is directly proportional to concentration, molar absorption coefficient, and optical path length
  • transmittance, absorbance, incident light, transmitted light
• To convert given absorbance back to transmittance:

  1 / antilog [absorbance)
• Protein must be at least 3000 daltons/3 kilodaltons to be detected by Bradford assay (1 dalton = 1g/mol)
• Absorbance measured with Bradford assay is linear up to 2000 ug/mL
• If absorbance of sample is higher than the absorbance of the standard solution with the highest concentration:

  1. sample should be diluted
  2. another standard solution with a higher concentration should be prepared
• Presence of surfactants/detergents in protein solution causes precipitation of CBB G-250, interfering with dye-protein complex formation
• Absorbance should be measured immediately (less than 1 hour) after mixing because the dye-protein complex also tends to precipitate
• Sensitivity of CBB G-250 varies from protein to protein. The standard curves made using the same concentrations of 2 different proteins will not be the same.
• In any protein assay, the best protein to use as a standard is a purified preparation of the protein being assayed.
• Bradford reagent must be stored away from light, as well as the incubation of protein solutions after mixing.
• R or r (given by spectrophotometer): correlation coefficient

  • measures strength and direction of linear relationship between two variables
  • ranges from -1 to 1
• R^2 or r^2 (given by MS Excel): coefficient of determination

  • quantifies proportion of the variance in the dependent variable (y) that is predicted or explained by independent variable (x)
  • ranges from 0 to 1