Protein Content Determination: Bradford Assay

• Protein Content Determination - Most protein assays take advantage of a reaction between a reagent dye and the protein of interest that will shift or increase the absorbance of a

  particular wavelength.
• Generally the more protein in a sample the higher

  the absorbance. BUT remember the assay must

  remain linear and within Beer’s Law!
• Beer's Law - states that the concentration of a chemical

  solution is directly proportional to its absorption of light. The premise is that a beam of light becomes weaker as it passes through a chemical solution.
• Equation for Beer's Law

  A = εbc

  where A is absorbance (no units)

  ε is the molar absorptivity with units of L mol-1 cm-1

  (formerly called the extinction coefficient)

  b is the path length of the sample, usually

  expressed in cm

  c is the concentration of the compound in solution,

  expressed in mol L-1
• Bradford Assay (Bradford, 1976)

  • Rapid
  • Easy
  • Performed at room temperature
  • Equipment requirement - spectrophotometer
• Basic Instrumentation of a Spectrophotometer

  Light source -> Collimator -> Monchromator (Prism of Grating) -> Slit (Wavelength selector) -> Cuvette (Sample Solution) -> Detection (Photocell) -> Digital Display
• Absorption Range

  220 to 280 nm - Aliphatic (or) alicyclic hydrocarbons (or) their derivatives

  220 to 250 nm - The compounds contain two unsaturated linkages in conjugation. Also, be due to “Benzene derivatives.”

  250 to 330 nm - The presence of more than two conjugated

  double bonds usually gives rise to absorption.

  450 to 500nm - Beta-carotene, a precursor of Vitamin A has

  eleven double bonds in a conjugated system and appears yellow.

  250 to 330 nm (249nm; 260nm and 325nm) - Vitamin K1 (Due to the presence of “NAPTHAQUINONE”)
• Principle of the Bradford Assay

  • Uses Coomassie G-250 dye, a colorimetric reagent for the detection and quantitation of total protein.
  • Acidic environment of the reagent, protein binds to the Coomassie dye.
  • Results in a spectral shift from the reddish/brown form of the dye (absorbance maximum at 465nm) to the blue form of the dye (absorbance maximum at 610 nm).
  • Optimal wavelength to measure the blue color from the Coomassie dye-protein complex is at 595nm.
• • Development of color in Coomassie dye-based (Bradford) protein assays has been associated with the presence of certain basic amino acids (primarily arginine, lysine and histidine) in the protein.
  • Van der Waals forces and hydrophobic interactions also participate in the binding of the dye by protein.
• The number of Coomassie dye ligands bound to each protein molecule is approximately proportional to the number of positive charges found on the protein.
• Free amino acids, peptides and low molecular weight proteins do not produce color with Coomassie dye reagents.
• In general, the mass of a peptide or protein must be at least 3000 daltons to be assayed with this reagent.
• The assay is performed at room temperature and

  no special equipment is required. Simply add the

  sample to the tube containing reagent and the

  resultant blue color is measured at 595nm

  following a short room-temperature incubation.
• Bradford reagent: Dissolve 100 mg Coomassie Brilliant Blue G-250 in 50 ml 95% ethanol, add 100 ml 85% (w/v) phosphoric acid. Dilute to 1 liter when the dye has completely dissolved,

  and filter through Whatman #1 paper just before use.
• The Bradford reagent should be a light brown in color. Filter repeatedly if blue components are visible. (Ready made Bradford reagents are available)
• Procedure- Assay

  1. Warm up the spectrophotometer before use.
  2. 2.Dilute unknowns if necessary to obtain between 5 and 100 μg protein in at least one assay tube containing 100 μl sample
  3. If desired, add an equal volume of 1 M NaOH to each sample and vortex (see Comments below). Add NaOH to standards as well if this option is used.
  4. Prepare standards containing a range of 5 to 100 micrograms protein (albumin or gamma globulin are recommended) in 100 μl volume.
  5. Add 5 ml dye reagent and incubate 5 min.
  6. Measure the absorbance at 595 nm.
• Procedure- Analysis
• Procedure- Analysis

  1. Prepare a standard curve of absorbance versus micrograms protein

  2. Determine amounts from the curve

  3. Determine concentrations of original samples from the amount protein, volume/sample, and dilution factor, if any
• Dye reagent

  Reacts primarily with arginine residues and less so with histidine, lysine, tyrosine, tryptophan, and phenylalanine residues
• Bradford assay

  Sensitive to bovine serum albumin, hence immunoglogin G (IgG - gamma globulin) is the preferred protein standard
• Addition of 1 M NaOH

  Suggested by Stoscheck (1990) to allow the solubilization of membrane proteins and reduce the protein-to-protein variation in color yield