Western blotting is a widely used technique for detecting and quantifying specific proteins within a complex mixture, from cell or tissue extracts to purified samples. The process involves several key steps that must be carefully optimized to ensure accurate and reproducible results.
General Western Blot Protocol
Based on recent detailed protocols, the core workflow includes the following steps:
- Sample Preparation: Cell or tissue lysates are prepared using appropriate lysis buffers containing protease inhibitors to prevent protein degradation. Protein concentration is measured (typically via BCA or Bradford assay) to load equal amounts of protein across samples.
- Gel Electrophoresis: Proteins are denatured by boiling in SDS-containing loading buffer, which coats proteins with a negative charge proportional to their size. They are then separated based on molecular weight via SDS-PAGE using polyacrylamide gels. The gel percentage depends on the target protein size, with higher percentages used for smaller proteins.
- Transfer to Membrane: Proteins are transferred from the gel onto a membrane such as nitrocellulose or PVDF. This involves assembling a transfer sandwich and applying an electric current in transfer buffer to facilitate migration.
- Blocking: The membrane is incubated in blocking solution to reduce nonspecific antibody binding.
- Antibody Incubation: The membrane is incubated first with a primary antibody specific to the target protein, followed by a secondary antibody conjugated to an enzyme such as HRP. Incubation times and dilutions depend on antibody affinity and sensitivity needs.
- Detection: Chemiluminescent substrates are used to visualize protein bands via film or digital imaging. Alternatively, fluorescently labeled antibodies allow multiplex detection using compatible imaging systems.
- Data Analysis: Band intensities are quantified using imaging software to compare relative protein expression levels. Proper blocking and washing reduce background and nonspecific signals.
