Makindo Medical Notes

Related Subjects: |Major Histocompatibility complex |Blotting Techniques: Gel Electrophoresis

Blotting 🧪 is a cornerstone technique in molecular biology for detecting specific macromolecules (DNA, RNA, or proteins). It usually follows gel electrophoresis ⚡, which separates molecules based on size and charge, making it possible to analyse individual fragments.

🔬 Introduction to Gel Electrophoresis

• Size & Mobility: ⚖️ Larger molecules move more slowly through the gel; smaller molecules travel further.
• Charge: ➕ Molecules with less negative charge migrate more slowly towards the positive electrode.
• Structure: 🌀 Secondary/tertiary folding can affect mobility. Denaturing agents (e.g. SDS for proteins, urea for nucleic acids) are used to unfold molecules so migration depends mainly on size.

📊 Blotting Techniques Overview

Different blotting methods are named by compass directions or analogy, each targeting a different macromolecule:

📌 Southern Blotting

Named after Dr. Edwin Southern. Detects specific DNA sequences.

• DNA is digested with restriction enzymes ✂️ into fragments.
• Separated by gel electrophoresis ⚡.
• Transferred to a membrane via capillary action 📜.
• Hybridised with labelled probes (radioactive, fluorescent, or enzymatic).
• Detected using autoradiography or chemiluminescence 🔦.

Clinical relevance: Used in early gene mapping, prenatal diagnosis, and identifying mutations (e.g. β-thalassaemia).

📌 Northern Blotting

Like Southern blotting but for RNA.

• Measures mRNA levels → shows which genes are switched on 🟢.
• Helpful in research and oncology (e.g. oncogene expression in cancers).

📌 Western Blotting

Detects specific proteins, widely used in research and diagnostics.

• Proteins separated by SDS-PAGE ➡️ transferred to PVDF/nitrocellulose membrane.
• Probed with antibodies 🎯 (primary antibody + labelled secondary antibody).
• Signal visualised with enzyme reactions (chemiluminescence) or fluorescence 🌟.
• Applications:
  • Confirmatory test for HIV infection (after ELISA).
  • Detecting Lyme disease.
  • Studying protein mutations (e.g. haemoglobin variants).

📌 Eastern & Southwestern Blots

• Eastern: Looks at how proteins are modified after synthesis (glycosylation, phosphorylation). Important in signalling and oncology.
• Southwestern: Explores protein–DNA binding → useful in transcription factor research.

💡 Key Clinical Takeaways

• 🧬 Southern → DNA mutation detection.
• 📜 Northern → Gene expression studies.
• 🧫 Western → Protein diagnostics (HIV, Lyme, CJD).
• ✨ Eastern → Post-translational modifications.
• 🔗 Southwestern → Protein–DNA interactions.

👉 Blotting techniques are **powerful bridges between molecular biology and clinical practice**. They underpin modern diagnostics, guide treatment decisions (e.g. HIV confirmation), and remain vital in research on cancer, genetics, and immunology.