Western Blotting Notes

Western Blotting

Western blotting (protein immunoblot) is a technique used to confirm the presence of a specific target protein in a sample.

SDS-PAGE

The process begins with SDS-PAGE (sodium dodecyl-sulfate polyacrylamide gel electrophoresis).

  • Treatment with SDS:
    • Proteins are denatured.
    • Proteins are coated with negative charges.
    • Proteins have a similar mass-to-charge ratio.
  • Gel electrophoresis:
    • Proteins migrate through the gel towards the positively charged anode.
    • Separation is based on molecular weight.
  • Staining:
    • Protein bands become visible after staining the gel.
  • Marker proteins:
    • Used as a reference to determine the molecular weight of proteins in the sample.
    • Example: A band may contain proteins with a molecular weight of approximately 60 kilodaltons.
  • Limitation of SDS-PAGE:
    • SDS-PAGE alone cannot identify the specific proteins present in a sample.
    • A band in the gel could be caused by the protein of interest or other proteins with a similar molecular weight.

Western Blot Procedure

1. Transfer
  • Proteins are transferred from the gel to a membrane (typically nitrocellulose).
  • Nitrocellulose membrane: Binds large amounts of protein.
  • Setup:
    • The gel is placed on top of the membrane.
    • The gel-membrane construct is flanked by filter papers on both sides.
    • All components are soaked in buffer to facilitate electricity transmission.
    • The setup is fixed between the cathode and the anode.
  • Process:
    • Electric current pulls the negatively charged proteins from the gel onto the membrane.
2. Blocking
  • The membrane is incubated with a blocking agent (milk or BSA - bovine serum albumin).
  • Purpose: To prevent antibodies from non-specifically binding to the membrane or other proteins.
3. Primary Antibody Incubation
  • The membrane is incubated with a primary antibody.
  • Specificity: The primary antibody is selected to bind exclusively to the protein of interest's epitopes.
4. Washing
  • Unbound primary antibodies are washed away.
  • Bound primary antibodies remain attached to the protein of interest.
5. Secondary Antibody Incubation
  • The membrane is incubated with a secondary antibody.
  • Specificity: The secondary antibody is specific for the primary antibody.
6. Washing
  • Unbound secondary antibodies are washed away.
  • Only secondary antibodies bound to the primary antibody remain.
7. Detection
  • The secondary antibody is conjugated to an enzyme (e.g., horseradish peroxidase - HRP).
  • Process:
    • When the membrane is incubated in a substrate mixture, the enzyme converts the substrate.
    • This conversion produces a chemiluminescent signal.
    • The chemiluminescent signal visualizes the protein band.
  • Result: The presence of the target protein is confirmed by the detection of the signal at the expected molecular weight.