Blotting Techniques & Electrophoresis
These are fundamental laboratory techniques used to separate, identify, and characterize nucleic acids and proteins. They are essential in both research and clinical diagnostics.
Electrophoresis
Separation of charged molecules in an electric field. Smaller and more charged molecules migrate faster. Support matrices: Agarose (for DNA/RNA), Polyacrylamide (PAGE, for proteins and small oligonucleotides).
- Agarose Gel Electrophoresis: Separates DNA/RNA by size. DNA stained with ethidium bromide (intercalates) or SYBR Green → visualized under UV. Smaller fragments → higher in gel if loading buffer used, lower mobility = larger fragments.
- SDS-PAGE (Sodium Dodecyl Sulfate-PAGE): SDS denatures proteins and imparts uniform negative charge proportional to mass → separation by SIZE ONLY. Molecular weight ladders used for comparison. Run under reducing conditions (DTT/β-mercaptoethanol to break disulfide bonds).
- Native PAGE: Proteins retain charge and shape → separated by charge, size, and shape. Used for isoenzyme separation (e.g., LDH, CK isoforms).
- 2D Electrophoresis: First dimension: Isoelectric focusing (IEF) separates by pI. Second dimension: SDS-PAGE separates by MW. Powerful for proteomics.
- Isoelectric Focusing (IEF): pH gradient in gel; protein migrates to its isoelectric point (net charge = 0) → stops. Used for Hb variants (HbS, HbF, HbA2).
Blotting Techniques — Mnemonic: SNoW DRoP
- Southern Blot (DNA): DNA extracted → restriction digest → agarose gel → denaturation → transfer to nitrocellulose membrane → hybridize with labeled probe (complementary DNA/RNA) → detect. Applications: RFLP analysis, gene mapping, detecting gene rearrangements (BCR-ABL in CML).
- Northern Blot (RNA): Same principle but for RNA. Shows gene expression level and mRNA size. Less common now (replaced by RT-qPCR).
- Western Blot (Protein): SDS-PAGE → transfer to PVDF membrane → block non-specific binding → probe with PRIMARY antibody → secondary antibody (HRP or fluorescent labeled) → detection. HIV diagnosis (confirmatory), clinical diagnostics. Distinguishes protein size and detects specific proteins.
- Southwestern Blot: DNA-binding proteins — protein blot probed with DNA.
- Far-Western Blot: Protein-protein interactions.
Dot Blot / Slot Blot
Quick immuno- or nucleic acid dot tests without gel electrophoresis. Used for rapid qualitative detection. Less information (no size data).
ELISA (Enzyme-Linked Immunosorbent Assay)
Antibody-based detection in microplate format. High sensitivity, high throughput. Types: Sandwich (capture + detection antibodies — quantitative), Competitive (inhibition-based), Direct, Indirect. Used for: HIV screening (p24 antigen or anti-HIV antibodies), pregnancy test (β-hCG), allergy testing, hormone levels.