Introduction
- Gel electrophoresis is one of the most fundamental techniques in molecular biology. It is widely used to separate DNA, RNA, and proteins based on their size and charge under the influence of an electric field.
What is Gel Electrophoresis?
Gel electrophoresis is a laboratory technique that separates molecules by forcing them to migrate through a gel matrix under an electric field. Larger molecules move slower, while smaller molecules move faster through the gel's pores.
Types of Gel Electrophoresis
|
Feature |
AGE (Agarose Gel Electrophoresis)
|
PAGE (Polyacrylamide Gel Electrophoresis) |
|
Gel material |
Agarose (From seaweed like Gracilaria and Gelidium |
Polyacrylamide |
|
Used for |
DNA & RNA |
Proteins |
|
Separation basis |
Molecular weight ( size) only |
Molecular weight and charge |
|
Pore size |
Larger, adjustable via concentration |
Smaller, precisely controlled |
Basis of Molecular Separation
DNA and RNA:
- DNA and RNA possess a uniformly negative charge because of their phosphate backbone.
- Therefore, they are separated mainly according to molecular size.
- Smaller fragments migrate faster, whereas larger fragments migrate more slowly.
Proteins:
- Proteins contain different amino acids and therefore possess different charges.
- Protein separation depends on both molecular weight and electrical charge.
Agarose Gel Mechanics & Buffer Chemistry
Pore Size vs Agarose Concentration
The agarose gel functions like a molecular sieve. The relationship between concentration and pore size is inversely proportional:
- High agarose concentration → smaller pore size → slower migration (ideal for separating small fragments)
- Low agarose concentration → larger pore size → faster migration (ideal for separating large fragments)
Role of Buffers: TAE and TBE
Buffers serve two critical functions during electrophoresis:
1. pH maintenance — they resist pH fluctuations that could damage the sample or affect migration.
2. Conductivity — they provide the electrolytic medium required for current flow.
|
Buffer |
Full Form |
|
TAE |
Tris, Acetate, EDTA |
|
TBE |
Tris, Borate, EDTA |
Why EDTA Matters
Many DNA/RNA-degrading enzymes (DNases and RNases) require magnesium ions (Mg²⁺) as cofactors to function. EDTA is a chelating agent that binds these Mg²⁺ ions, effectively deactivating the enzymes and protecting nucleic acid samples from degradation.
Visualization and Specialized Techniques
- EtBr (Ethidium Bromide): An intercalating agent that inserts itself between DNA base pairs and fluoresces under UV light, allowing DNA bands to be visualized. It is a known carcinogen and requires careful handling with gloves and UV protection.
- Sample Loading Dye: Substances like Bromophenol Blue are added to the sample to (a) track the progress of the run visually and (b) increase sample density so it sinks properly into the well.
- Isoelectric Focusing (IEF): A protein separation technique based on the isoelectric point (pI) — the pH at which a protein's net charge is zero. Proteins migrate through a pH gradient until reaching their specific pI, where migration stops and the protein "focuses."
SDS-PAGE: Mechanism and Composition
SDS-PAGE (Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis) is the standard method for separating proteins by molecular weight.
Resolving Gel vs Stacking Gel
|
Parameter |
Stacking Gel |
Resolving (Running) Gel |
|
Acrylamide concentration |
Low |
High |
|
Pore size |
Large |
Small |
|
pH |
6.8 |
8.8 |
Function : It concentrates proteins into a sharp band separates proteins by size
Key Components
- SDS (Sodium Dodecyl Sulfate): It denatures proteins and coats them with a uniform negative charge, ensuring separation is based purely on size, not native charge.
- APS (Ammonium Persulfate): It generates free radicals that initiate gel polymerization.
- TEMED: It acts as a catalyst, accelerating the polymerization reaction.
Running Buffer and Sample Preparation
- Running buffer: Tris-Glycine, pH 8.3
- Sample preparation mix: SDS, Bromophenol Blue, β-Mercaptoethanol (β-ME), and Tris-HCl (pH 6.8)
- β-ME breaks disulfide bonds, ensuring complete protein denaturation.
Glycine and the Isoelectric Point (pI) Concept
- Glycine, with a pI of approximately 5.8, is central to how the Tris-Glycine buffer system works during SDS-PAGE. Its charge shifts depending on the surrounding pH:
- At low pH (2.8): Glycine is protonated, carrying a positive charge.
- At high pH (9.6): Glycine is deprotonated, carrying a negative charge.
- This pH-dependent charge shift allows glycine ions to move ahead of proteins in the stacking gel and then slow down in the resolving gel, creating the sharp band resolution characteristic of SDS-PAGE.
Conclusion
Gel electrophoresis is one of the most important techniques in molecular biology for separating DNA, RNA, and proteins. Agarose Gel Electrophoresis (AGE) is mainly used for nucleic acids, while Polyacrylamide Gel Electrophoresis (PAGE), especially SDS-PAGE, is widely used for protein analysis.