Introduction
Western blotting is a technique used to detect specific proteins in a sample based on antigen-antibody interactions.
Steps in Immunoblotting
1) Protein Separation (Electrophoresis)
Proteins are separated by size and charge using polyacrylamide gel electrophoresis (PAGE).
2) Transfer to Membrane
Separated proteins are transferred onto a nitrocellulose or PVDF membrane for analysis.
3) Antibody Binding
A primary antibody binds to the target protein.
It may be labeled with a detection molecule (e.g., fluorescent dye, enzyme, or radioactive isotope).
4) Signal Detection
The detection molecule produces a signal visible via chemiluminescence, fluorescence, or radioactive imaging.
Signal intensity correlates with the protein amount.
5) Signal Amplification (Optional)
A secondary antibody binds to the primary antibody, enhancing detection sensitivity.
