Electrophoresis and Applications

Principles of Electrophoresis - Charge & Go!

• Separates charged molecules by differential migration in an electric field ($E$).
• Migration velocity ($v$): $v = \frac{Eq}{f}$
  • $E$: Electric field strength
  • $q$: Net molecular charge
  • $f$: Frictional coefficient (depends on size, shape, medium viscosity)
• Electrophoretic mobility ($\mu$): $\mu = \frac{v}{E} = \frac{q}{f}$. Intrinsic property.
• Key factors influencing speed & direction:
  • Net charge ($q$): ↑ magnitude $\rightarrow$ ↑ speed. Sign determines direction.
  • Size/Shape (via $f$): ↓ size/more compact $\rightarrow$ ↑ speed.
  • Electric field ($E$): ↑ $E$ $\rightarrow$ ↑ speed.
  • Support medium: Viscosity, pore size. , buffer, support medium with sample wells, and direction of ion movement)

⭐ Anions $\rightarrow$ Anode (+); Cations $\rightarrow$ Cathode (-). 📌 PANIC: Positive Anode, Negative Is Cathode (electrode polarity).

Gel Electrophoresis Types - Matrix Masters

• Agarose Gel Electrophoresis (AGE)
  • Agarose matrix (natural); large, adjustable pores (0.5-2% concentration).
  • Separates nucleic acids (DNA > 50 bp, RNA) primarily by size.
  • Uses: DNA fingerprinting, PCR product analysis, restriction mapping.
  • Stains: Ethidium Bromide (EtBr), SYBR Safe.
  • 📌 "AGE for large DNA, like sifting sand."
• Polyacrylamide Gel Electrophoresis (PAGE)
  • Polyacrylamide matrix (synthetic); small, uniform, adjustable pores (controlled by %T & %C).
  • Separates proteins and small DNA/RNA fragments (< 500 bp).
  • Types:
    • Native PAGE: Separates by charge, size, and shape. Proteins remain active.
    • SDS-PAGE: Sodium Dodecyl Sulfate (SDS) denatures proteins, imparts uniform negative (-) charge. Separates by molecular weight. Reducing agents (e.g., β-mercaptoethanol) break disulfide (S-S) bonds.
  • Stains: Coomassie Brilliant Blue, Silver stain (for proteins).

  ⭐ SDS-PAGE is the most common method for determining the molecular weight of proteins. oka

Advanced & Blotting Techniques - Focus & Find

• Advanced Electrophoresis:

  • Isoelectric Focusing (IEF): Separates proteins by isoelectric point (pI) using a pH gradient. Proteins migrate until net charge is zero.
  • 2D Gel Electrophoresis: Combines IEF (1st dimension: pI) with SDS-PAGE (2nd dimension: mass). High resolution for complex mixtures (e.g., proteomics).
  • Capillary Electrophoresis (CE): Automated, high-throughput, high-resolution separation in narrow fused-silica capillaries.

• Blotting Techniques: Transfer of separated biomolecules (DNA, RNA, or proteins) from a gel to a solid membrane for specific detection.

*   **Southern Blot:** Detects specific DNA sequences. Probe: Labeled DNA/RNA.
*   **Northern Blot:** Detects specific RNA sequences (gene expression). Probe: Labeled DNA/RNA.
*   **Western Blot:** Detects specific proteins. Probe: Labeled antibody.
    > ⭐ Western Blot is the confirmatory test for HIV, detecting antibodies to viral proteins (e.g., p24, gp41, gp120/160).
*   **Southwestern Blot:** Identifies DNA-binding proteins. Probe: Labeled DNA oligonucleotides.
*   **Far-Western Blot:** Detects protein-protein interactions. Probe: Labeled non-antibody protein.
*   **Eastern Blot:** Analyzes post-translational modifications (PTMs) of proteins.
*   📌 Mnemonic: **SNoW DRoP** (Southern=DNA, Northern=RNA, Western=Protein).

Clinical Applications - Health Detectives

• Serum Protein Electrophoresis (SPEP):
  • Key for monoclonal gammopathies (Multiple Myeloma: M-spike; MGUS).
  • Patterns reveal: inflammation (↑ acute phase reactants), nephrotic syndrome (↓ albumin, ↑ α2), cirrhosis (β-γ bridge).
• Hemoglobin Electrophoresis:
  • Diagnoses hemoglobinopathies: Sickle cell (HbS), β-thalassemia (↑HbA2/HbF).
• Lipoprotein Electrophoresis:
  • Classifies dyslipidemias (e.g., Fredrickson types).
• Isoenzyme Analysis:
  • CK-MB for MI; LDH patterns for organ-specific damage.
• Nucleic Acid Electrophoresis:
  • Genetic diagnosis (e.g., mutations), viral detection.

⭐ The M-spike on SPEP, a sharp peak usually in the γ-globulin region, is a hallmark of Multiple Myeloma.

High‑Yield Points - ⚡ Biggest Takeaways

• Electrophoresis separates molecules by charge, size, and shape in an electric field.
• SDS-PAGE separates proteins by molecular weight; SDS imparts uniform negative charge.
• Agarose gels are used for DNA/RNA separation; polyacrylamide for proteins.
• Isoelectric focusing (IEF) separates proteins based on their isoelectric point (pI).
• 2D electrophoresis combines IEF and SDS-PAGE for superior protein resolution.
• Key blotting techniques: Southern (DNA), Northern (RNA), Western (Protein) (SNoW DRoP).
• Diagnoses hemoglobinopathies, paraproteinemias, and genetic disorders.