Protein Immunoblotting (Western Blot)

Overview

• Technique used to confirm the presence of a specific target protein in a sample.
• Follows initial separation using SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis).

SDS-PAGE Process

• Denaturation: Proteins in the sample are treated with SDS, causing them to denature and acquire negative charges.
• Separation: Proteins travel through the gel toward the positively charged anode, separating based on molecular weight.
• Staining: After electrophoresis, the gel is stained to visualize protein bands.
• Marker Proteins: Reference proteins are used to determine the molecular weight of proteins in the sample.
  • Example: A band observed at approximately 60 kilodaltons.

Limitations of SDS-PAGE

• SDS-PAGE only indicates the presence of proteins at specific molecular weights; it does not confirm identity.
  • Protein bands could potentially include various proteins, not just the target of interest.

Western Blot Process

Setup

• Gel Transfer: The gel containing separated proteins is placed on a nitrocellulose membrane, which binds proteins.
• Electric Current: The gel-membrane assembly is placed between cathode and anode, allowing proteins to transfer to the membrane due to electric current.

Incubation Steps

1. Blocking:

   • Membrane incubated with milk or BSA (Bovine Serum Albumin) to prevent nonspecific binding of antibodies.

2. Primary Antibody Incubation:

   • Membrane incubated with a primary antibody that specifically binds the target protein.

3. Washing:

   • Wash away unbound primary antibodies without removing those bound to the target protein.

4. Secondary Antibody Incubation:

   • Incubation with a secondary antibody that is specific to the primary antibody.

5. Final Washing:

   • Remove unbound secondary antibodies; only those bound to the primary remain.

Protein Detection

• Enzyme Conjugation: Secondary antibodies are often conjugated with an enzyme (e.g., horseradish peroxidase).
• Chemiluminescent Reaction: When incubated in a substrate mixture, the enzyme converts the substrate to produce a chemiluminescent signal.
• Visualization: This signal visualizes the protein band, confirming the presence of the target protein.

Conclusion

• Western blotting is a powerful technique for confirming protein presence in samples.
• Importance of each step in ensuring specificity and accuracy in protein detection.