Western blotting is a widely used technique for detecting and analyzing specific proteins in a sample.
Principle
Western blotting is based on antigen-antibody interaction, where a specific antibody binds to the target protein for detection.
Steps of Western Blotting
Sample Preparation: Proteins are extracted from cells or tissues and denatured using SDS (Sodium Dodecyl Sulfate).
Gel Electrophoresis (SDS-PAGE): Proteins are separated based on their molecular weight in a polyacrylamide gel.
Transfer to Membrane: The proteins are transferred from the gel to a membrane (nitrocellulose or PVDF) using an electric field.
Blocking: The membrane is blocked with a protein solution (e.g., BSA or milk) to prevent non-specific antibody binding.
Incubation with Primary Antibody: A specific antibody binds to the target protein.
Incubation with Secondary Antibody: A secondary antibody conjugated with an enzyme (e.g., HRP) binds to the primary antibody.
Detection: A chemiluminescent or colorimetric substrate is added, and the signal is visualized using X-ray film or a digital imaging system.
Types of Western Blotting
Two-dimensional (2D) Western blotting
Multiplex Western blotting
Applications of Western Blotting
Protein expression analysis
Protein-protein interaction analysis
Post-translational modification analysis
Disease diagnosis
