Experiment 6: Protein Quantification Assay

Experiment 6: Quantitative Determination of Protein by a Dye Binding Assay

• Aim: To determine the protein concentration and purity of an enzyme.
• Procedure:
  • Prepare a standard curve using Coomassie Brilliant Blue G-250 dye binding assay.
  • Determine the protein concentration of mitochondrial suspensions from plant and liver.
  • Calculate the specific activity of succinate dehydrogenase (SDH) in each mitochondrial suspension.
  • Compare the specific activity values of SDH from different sources (plant/animal cells).

Introduction

• Protein concentration determination is crucial in enzyme purification.
• The Bradford assay (dye-binding assay) uses Coomassie Brilliant Blue G-250 dye binding to proteins in phosphoric acid.
• Two forms of the dye are shown (Coomassie Brilliant Blue G and Coomassie Brilliant Blue R). A key difference is the addition of two methyl groups in Coomassie Brilliant Blue R-250 making it unsuitable for this experiment.
• The Bradford assay is rapid, sensitive, and easy to perform; minimal interference from other components.
• The method is accurate for protein concentration, from 5-40µg, as low as 1µg in the sample solution.
• Important note: Free amino acids and small peptides do not react with the dye. This makes it applicable to crude samples without dialysis.

Principle of Bradford Assay

• Coomassie Brilliant Blue G-250 dye (CBB dye) binds non-covalently to proteins in an acidic solution.
• The binding of the dye to the protein changes the maximum absorbance wavelength from 465nm for free dye, to 595nm.
• The increase in absorbance (A) at 595nm is directly proportional to the protein concentration in the solution.

Advantages of Bradford Assay

• Simple to perform: Requires just one reagent.
• Rapid: Color development is complete within ~2 minutes.
• Sensitive: Allows for the measurement of low protein concentrations.
• Low interference: Minimal interference from non-protein components like ammonium sulfate, polyphenols, sucrose, and cations (e.g., K+, Na+, Mg2+).
• Can also be used in presence of chelating agents (e.g., EDTA) and reducing agents (e.g. mercaptoethanol).

Disadvantages of Bradford Assay

• Interference from detergents: Low concentrations of non-ionic Triton X-100 increase assay sensitivity towards proteins; while anionic detergents (e.g., SDS) decrease absorbance at 595 nm.
• pH control: Alkaline protein samples may affect assay pH.
• Destructive to protein samples: Cannot recover the protein samples after the assay.
• Discoloration of glassware: The protein-dye complex may bind to glass and plastic, potentially staining them and affecting spectrophotometric readings

Preparation of the dye solution

• Weigh 120 mg of Coomassie Brilliant Blue G-250, add 100 ml of 85% phosphoric acid at 30 °C, and 50 ml of 95% ethanol to a beaker.
• Allow the dye to dissolve and mix for at least two hours with magnetic stirring.
• Bring the final volume to 1000 ml using distilled water and continue mixing for an additional hour.
• Filter the solution.
• Store the solution in dark bottles and store it at room temperature.

Procedure for Dilutions (Standard Curve)

• Label six test tubes.
• Using a micropipette, deliver decreasing volumes of distilled water into tubes 1 to 6.
• Use the same micropipette tip to deliver increasing volumes of the standard protein (BSA) solution to give increasing concentrations in each tube.
• The total volume in each tube should be 1ml.
• Cover each tube with Parafilm® and mix by inverting
• Note the procedure for preparing a standard curve
• Note the procedure for applying the dye solution to various samples for measuring absorbance, using a spectrophotometer.

Determining Protein Concentration of Samples

• Prepare diluted mitochondrial suspensions.
• Use the standard curve to determine protein concentration from sample absorbance

Calculating Specific Activity

• Calculate the specific activity of Succinate Dehydrogenase (SDH) using the following formula:
• Specific activity = SDH activity (mIU/ml of MS) / mg protein/ml of MS.

This quiz covers the quantitative determination of protein concentration using the Coomassie Brilliant Blue G-250 dye binding assay. You'll learn about the procedure for preparing a standard curve, measuring protein concentration in different sources, and calculating the specific activity of enzymes like succinate dehydrogenase. Understanding these concepts is essential for enzyme purification and analysis.