Gel Electrophoresis Techniques

Gel Electrophoresis

• Purpose: Separate proteins based on size and charge
• Types of gels:
  • SDS-PAGE (Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis): Most commonly used for Western Blot
  • Native PAGE: For native proteins, without SDS
• Process:
  1. Sample preparation: Mix protein sample with sample buffer (SDS, glycerol, and bromophenol blue)
  2. Load samples into gel wells
  3. Apply electric current: Proteins migrate through the gel based on size and charge
  4. Stop the run: When the dye front reaches the end of the gel

Protein Extraction

• Purpose: Isolate proteins from cells or tissues
• Methods:
  • Mechanical disruption (e.g., sonication, homogenization)
  • Enzymatic disruption (e.g., lysozyme)
  • Detergent-based extraction (e.g., Triton X-100)
• Considerations:
  • Sample type (e.g., cells, tissues, biofluids)
  • Protein solubility and stability
  • Avoid protein degradation and contamination

Antibody Selection

• Purpose: Detect specific proteins of interest
• Types of antibodies:
  • Primary antibodies: Recognize specific epitopes on the target protein
  • Secondary antibodies: Bind to primary antibodies and facilitate detection
• Considerations:
  • Antibody specificity and sensitivity
  • Host species and isotype (e.g., rabbit IgG, mouse IgM)
  • Cross-reactivity and non-specific binding

Membrane Transfer

• Purpose: Transfer separated proteins from the gel to a membrane
• Methods:
  • Electroblotting (wet or semi-dry transfer)
  • Capillary transfer (dry transfer)
• Considerations:
  • Membrane type (e.g., nitrocellulose, PVDF)
  • Transfer buffer composition and pH
  • Transfer time and voltage

Protein Detection

• Purpose: Visualize bound antibodies and detect target proteins
• Methods:
  • Chemiluminescence (e.g., ECL, SuperSignal)
  • Fluorescence (e.g., FITC, Alexa Fluor)
  • Chromogenic (e.g., TMB, DAB)
• Considerations:
  • Detection sensitivity and specificity
  • Background signal reduction (e.g., blocking, washing)
  • Signal amplification (e.g., tyramide signal amplification)

Gel Electrophoresis

• Separates proteins based on size and charge
• Two types of gels: SDS-PAGE (most commonly used for Western Blot) and Native PAGE (for native proteins without SDS)
• Process involves:
  • Sample preparation: mixing protein sample with sample buffer (SDS, glycerol, and bromophenol blue)
  • Loading samples into gel wells
  • Applying electric current: proteins migrate through the gel based on size and charge
  • Stopping the run: when the dye front reaches the end of the gel

Protein Extraction

• Isolates proteins from cells or tissues
• Three methods: Mechanical disruption (e.g., sonication, homogenization), Enzymatic disruption (e.g., lysozyme), and Detergent-based extraction (e.g., Triton X-100)
• Considerations:
  • Sample type (e.g., cells, tissues, biofluids)
  • Protein solubility and stability
  • Avoiding protein degradation and contamination

Antibody Selection

• Detects specific proteins of interest
• Two types of antibodies:
  • Primary antibodies: recognize specific epitopes on the target protein
  • Secondary antibodies: bind to primary antibodies and facilitate detection
• Considerations:
  • Antibody specificity and sensitivity
  • Host species and isotype (e.g., rabbit IgG, mouse IgM)
  • Cross-reactivity and non-specific binding

Membrane Transfer

• Transfers separated proteins from the gel to a membrane
• Two methods: Electroblotting (wet or semi-dry transfer) and Capillary transfer (dry transfer)
• Considerations:
  • Membrane type (e.g., nitrocellulose, PVDF)
  • Transfer buffer composition and pH
  • Transfer time and voltage

Protein Detection

• Visualizes bound antibodies and detects target proteins
• Three methods:
  • Chemiluminescence (e.g., ECL, SuperSignal)
  • Fluorescence (e.g., FITC, Alexa Fluor)
  • Chromogenic (e.g., TMB, DAB)
• Considerations:
  • Detection sensitivity and specificity
  • Background signal reduction (e.g., blocking, washing)
  • Signal amplification (e.g., tyramide signal amplification)

Western Blot Overview

• A laboratory technique that detects and analyzes specific proteins in a sample using electrophoresis and immunodetection principles.
• Also known as immunoblotting or protein blotting.

Steps Involved in Western Blot

• Sample preparation: Protein samples are extracted from cells or tissues and denatured to create a linear sequence of amino acids.
• Electrophoresis: Denatured proteins are separated based on their molecular weight using SDS-PAGE.
• Transfer: Separated proteins are transferred from the gel to a membrane (nitrocellulose or PVDF) using an electric current.
• Blocking: The membrane is treated with a blocking agent to prevent non-specific binding of antibodies.
• Antibody incubation: The membrane is incubated with a primary antibody that specifically binds to the target protein.
• Detection: The primary antibody is detected using a secondary antibody conjugated to an enzyme (e.g., HRP) or a fluorophore.
• Visualization: The enzyme or fluorophore is used to visualize the protein bands on the membrane.

Types of Western Blot

• Monoclonal Western Blot: Uses a monoclonal antibody that recognizes a specific epitope on the target protein.
• Polyclonal Western Blot: Uses a polyclonal antibody that recognizes multiple epitopes on the target protein.
• Far-Western Blot: Uses a protein-protein interaction to detect the target protein.

Applications of Western Blot

• Protein expression analysis: Studies the expression levels of specific proteins in different cell types or tissues.
• Protein modification analysis: Studies post-translational modifications (e.g., phosphorylation, ubiquitination) of proteins.
• Biomarker detection: Identifies and quantifies specific proteins as biomarkers for diseases or conditions.
• Quality control: Validates the presence and purity of recombinant proteins in biotechnology applications.