Laboratory Techniques in Protein Analysis

Laboratory Week 12 - SDS-PAGE and Bradford

Overview of Laboratory Exercises (Weeks 10-13)

  • Key Experiments:
    • Protein expression
    • Protein purification
    • SDS-PAGE and protein concentration
    • Western blot

Protein Characterization Techniques

  1. SDS-PAGE
    • Stands for Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis.
    • Differences from agarose gels are significant; SDS-PAGE is specific for proteins.
  2. Methods to Calculate Protein Concentration:
    • Bradford assay
    • BCA (Bicinchoninic Acid)
    • UV absorbance at 280 nm

Understanding SDS-PAGE

  • Components:
    • Sodium Dodecyl Sulfate (SDS): A detergent that binds proteins, imparting a negative charge.
    • Polyacrylamide gel: The medium that separates proteins based on size.
  • Mechanism:
    • Small proteins move more quickly through the gel, while larger proteins move more slowly.
    • SDS binds to proteins stoichiometrically, ensuring that they all have a similar charge-to-mass ratio, which is crucial for separation.

Steps to Perform SDS-PAGE

  1. Preparation:
    • Obtain purified protein.
    • Add SDS to the protein sample, which covers the proteins with a negative charge.
    • Add β-mercaptoethanol (β-mer), which denatures proteins by breaking disulfide bonds, allowing them to unfold.
  2. Loading and Running the Gel:
    • Load the prepared protein mixture (with SDS and a dye) onto the polyacrylamide gel.
    • Apply an electric current to the gel; smaller proteins will migrate faster towards the anode.
  3. Visualizing Proteins:
    • After electrophoresis, staining of the gel is required to visualize proteins.
    • A common dye used is Coomassie Brilliant Blue, which specifically stains proteins.
    • Destaining process: Necessary to remove non-specific dye from the gel, allowing for clearer visualization of proteins.

Additional Steps in SDS-PAGE

  • Gel Assembly: The gel must be assembled in a gel box and must include 200 mL of polyacrylamide gel solution.
  • Transferring Proteins for Western Blot:
    • A buffer-soaked filter paper is placed on both sides of the polyacrylamide gel during transfer.
    • Transfer occurs via an electric field, directing protein migration to the membrane.

Bradford Assay for Protein Concentration Determination

  1. Principle:
    • Involves adding Coomassie Brilliant Blue dye to the protein sample in acidic conditions.
    • Upon binding to the protein, the dye’s absorbance shifts from 465 nm (unbound) to 595 nm (bound). This shift indicates the presence of protein.
    • The greater the protein concentration, the higher the absorbance at 595 nm.
  2. Creating a Calibration Curve:
    • Use Bovine Serum Albumin (BSA) as a standard for known protein concentrations.
    • Plot A595 against the micrograms of BSA for the standard curve, determining the concentration of unknown samples by comparing their A595 values.
    • Note: A new calibration curve must be established each time the Bradford assay is performed to ensure accuracy in measurement.

Important Notes

  • SDS-PAGE is significantly different from agarose gel, as it is specifically used for separating proteins rather than nucleic acids.
  • Understanding the underlying mechanisms of SDS-PAGE and the Bradford assay is crucial for accurate protein characterization in biochemical research and applications.