Makindo Medical Notes | 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:

🧾 Blot Type	🎯 Target Molecule	🔍 Key Use
Southern Blot	DNA 🧬	Detects specific DNA sequences → used in genetic mapping, mutations (e.g. sickle cell).
Northern Blot	RNA 📜	Analyses gene expression → shows mRNA levels in different tissues.
Western Blot	Proteins 🧫	Identifies proteins with antibodies → diagnostic tool (e.g. HIV, Lyme disease).
Eastern Blot	Protein Modifications ✨	Detects post-translational changes → e.g. phosphorylation, glycosylation.
Southwestern Blot	Protein–DNA interactions 🔗	Studies transcription factor binding and gene regulation.

📌 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.

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Blotting Techniques: Gel Electrophoresis