Western Blotting

Western Blotting

Principle

Western blotting detects specific proteins in a sample using a combination of gel electrophoresis, transfer to a membrane, and antibody-based detection. It relies on the specificity of antibodies to target proteins, enabling quantitative or qualitative analysis.

Instrumentation

  1. SDS-PAGE Apparatus: For separating proteins based on molecular weight.
  2. Blotting Apparatus: Transfers proteins from gel to membrane (electroblotting or semi-dry blotting systems).
  3. Membrane: Nitrocellulose or polyvinylidene difluoride (PVDF) for protein immobilization.
  4. Blocking and Hybridization Setup: Trays or incubators for applying blocking agents, primary antibodies, and secondary antibodies.
  5. Detection Systems:
    • Chemiluminescent imaging systems.
    • Colorimetric detection.
    • Fluorescence-based detection.

Types of Western Blotting

  1. Conventional Western Blotting: Uses chemiluminescent or colorimetric detection systems with enzyme-linked antibodies.
  2. Fluorescent Western Blotting: Utilizes fluorescently labeled antibodies for simultaneous detection of multiple proteins.
  3. Quantitative Western Blotting: Measures protein levels accurately using densitometry.
  4. Reverse Western Blotting: Probes specific proteins against a library of antibodies.

Applications of Western Blotting

  1. Protein Identification: Detecting specific proteins in complex samples.
  2. Post-Translational Modifications: Analyzing phosphorylation, glycosylation, or ubiquitination.
  3. Disease Biomarker Detection: Identifying proteins associated with diseases (e.g., cancer, viral infections).
  4. Gene Expression Validation: Confirming the presence of proteins encoded by expressed genes.
  5. Antibody Validation: Testing the specificity and performance of antibodies.
  6. Drug Development: Studying protein-drug interactions.
  7. Forensic Analysis: Detecting human proteins for forensic identification.
  8. Quality Control in Biomanufacturing: Ensuring the purity and identity of protein products.




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