Molecular Biology Techniques: Blotting, Hybridoma, and Fermentation

Posted on May 23, 2026 in Biotechnology

Immunoblotting

Viral antigens are detected with a polyclonal or a monoclonal antibody (MAb) on nitrocellulose paper.

  • After incubation, the protein bands (immune complexes) are visualized with peroxidase-conjugated protein and a color reagent.
  • A color develops in the bands where the antibody binds to the antigen.
  • Immunoblotting is an assay mixture of these two techniques.

Western Blotting

Western blotting is based on the principles of immunochromatography where proteins are separated in polyacrylamide gel according to their isoelectric point and molecular weight. It is a technique for detecting specific proteins separated by electrophoresis using labeled antibodies.

Immunoblotting is performed chiefly in diagnostic laboratories to identify desirable protein antigens in complex mixtures. An improved immunoblot method, Zestern analysis, addresses this issue without the electrophoresis step, significantly improving the efficiency of protein analysis.

Related Techniques

Other related techniques include dot blot analysis, immunohistochemistry (where antibodies detect proteins in tissues and cells by immunostaining), and Enzyme-Linked Immunosorbent Assay (ELISA).

Enzyme-Linked Immunosorbent Assay (ELISA)

ELISA is a rapid immunochemical test involving an enzyme that catalyzes a biochemical reaction, along with an antibody or antigen. ELISA tests detect substances with antigenic properties, primarily proteins, such as hormones, bacterial antigens, and antibodies.

Types of ELISA

  1. Direct ELISA
  2. Indirect ELISA
  3. Sandwich ELISA
  4. Competitive ELISA

Applications of ELISA

  • Determining the presence of antibodies and antigens in a sample.
  • Detecting food allergens in the food industry.
  • Determining the concentration of serum antibody in a virus test.
  • Evaluating disease spread during outbreaks (e.g., COVID-19).

Western Blotting Procedure

  1. Gel Electrophoresis: Protein samples are separated using SDS-PAGE.
  2. Transfer: Separated proteins are transferred to a solid support like a nitrocellulose or polyvinylidene difluoride membrane.
  3. Total Protein Staining: Proteins are visualized using dyes such as Ponceau S, Coomassie R-350, or amido black.
  4. Blocking: Prevents non-specific interaction between the membrane and the antibody.
  5. Incubation: An antibody with a reporter enzyme is probed on the membrane.
  6. Detection and Visualization: Unbound probes are washed away, and a colorimetric reaction is produced.

Applications

  • Detection of specific proteins from a mixture.
  • Size and amount estimation of proteins.
  • Verification following high-sensitivity ELISA for HIV, BSE, and HBV diagnosis.
  • Detection of condensed protein isoforms and tagged proteins.

Southern Blotting

Southern blotting is a molecular biology technique used for DNA detection, characterization, and quantification. It is an analytical technique for identifying specific DNA sequences by separating fragments on a gel and transferring them to a carrier membrane for hybridization.

Procedure

  1. DNA Extraction: DNA is purified from cellular material using detergent lysis and alcohol precipitation.
  2. DNA Fragmentation: Restriction endonuclease enzymes break long sequences into smaller fragments.
  3. Gel Electrophoresis: DNA fragments are sorted by size using agarose or polyacrylamide gels.
  4. Denaturation: Alkalis convert double-stranded DNA to single-stranded DNA.
  5. Blotting: DNA is transferred to a solid support and fixed via heat or UV radiation.
  6. Washing: Buffers remove excess probes.

Hybridoma Technology

Hybridoma technology produces large quantities of monoclonal antibodies (mAb) with single specificity by fusing a spleen cell (B-cell) with a myeloma cell.

Procedure

  1. Immunization: An antigen is injected into an animal to activate B-cells.
  2. Isolation: Myeloma cells (immortal cancerous cells) are isolated from bone marrow.
  3. Fusion: Spleen cells and myeloma cells are fused using PEG or electro-fusion.
  4. Selection: HAT medium (Hypoxanthine, Aminopterin, Thymidine) is used to select hybrid cells.
  5. Screening: ELISA is used to identify positive hybridoma cultures.

Fermentation

Fermentation is the chemical transformation of organic substances into simpler compounds by enzymes produced by microorganisms like molds, yeasts, or bacteria.

Types of Fermentation

  • Aerobic: Requires oxygen (e.g., wine, beer, vinegar).
  • Anaerobic: Extracts energy from carbohydrates without oxygen.
  • Homo-Lactic: Produces only lactic acid.
  • Hetero-Lactic: Produces lactic acid and gaseous by-products.

Food Preservation

Fermentation (lacto-fermentation) breaks down starches and sugars, acting as a natural preservative while increasing digestibility and beneficial enzymes.

Advantages and Disadvantages

  • Advantages: Produces lactic acid, generates ATP, and preserves food.
  • Disadvantages: Slow production, impure products requiring further treatment, and high energy costs.