What is Western Blot?
Western Blot (Protein Immunoblot) is a widely used method for protein detection and identification of specific proteins in protein solution in areas such as protein biology, immunogenetics, and molecular biology. Western blot technique combines the specificity of antibody detection with the resolution of gel electrophoresis.
Western Blot Methods
- Sample Preparation
- Gel electrophoresis
- Transfer of proteins to the membrane
- Antibody incubation
- Imaging and analysis
How is Western Blot applied?
The first step in the Western blot procedure is to prepare the sample. Next, macromolecules in a sample are separated using gel electrophoresis. The separated molecules are then transferred or blotted onto a second matrix, usually a nitrocellulose or polyvinylidene difluoride (PVDF) membrane. The membrane is then blocked to prevent any non-specific binding of antibodies to the surface of the membrane.
Most commonly, the transferred protein is then probed with a combination of antibodies: an antibody specific to the protein of interest (primary antibody) and another antibody specific to the host type of the primary antibody (secondary antibody). Usually, the secondary antibody complexes with an enzyme which, when combined with a suitable substrate, will produce a detectable signal. Chromogenic substrates produce a precipitate that results in visible colorimetric changes on the membrane.
The most sensitive detection methods use a chemiluminescent substrate that produces light as a byproduct of the reaction with the enzyme conjugated to the antibody. Light output can be captured using film. However, digital imaging devices based on charge-coupled device (CCD) cameras are becoming popular alternatives to film for capturing the chemiluminescent signal.
