Electrophoresis may look simple from the outside. Just bands moving across a gel. But the instrumentation behind it ensures precision, reproducibility, and safety.
Let’s break it down.
Agarose Gel
Agarose is a polysaccharide composed of:
- D-galactose
- 3,6-anhydro-L-galactose
Preparation:
- Agarose powder is dissolved in an appropriate buffer.
- Heated until the solution becomes clear.
- Poured into a casting tray with combs in place.
- After cooling and solidification, combs are removed– wells are formed.
Pore Size
Pore size depends on agarose concentration:
- Lower concentration —Larger pores
- Higher concentration —Smaller pores
- Typical range: 0.8% – 5%
Used mainly for:
- DNA
- RNA (formaldehyde is added while preparing the gel. It destroys any DNA present in the sample so you gel pure RNA bands)
- Large fragments
Polyacrylamide Gel Electrophoresis (PAGE) – Instrumentation
Polyacrylamide gels provide higher resolution than agarose gels and are ideal for proteins and small DNA fragments.
Components of the Gel:
1. Acrylamide
- Main monomer
- Forms long polymer chains
2. Bisacrylamide
- Cross-linking agent
- Creates a mesh-like structure
3. Ammonium Persulfate (APS)
- Generates free radicals
- Initiates polymerization
4. TEMED
- Catalyst
- Accelerates free radical formation from APS
- Speeds up polymerization
5. Buffer
- Maintains stable pH
- Preserves protein structure (unless denaturing conditions are used)
SDS-PAGE (Denaturing PAGE)
When separating proteins by size:
6. Sodium Dodecyl Sulfate (SDS)
- An anionic detergent
- Denatures proteins
- Imparts a uniform negative charge
- Ensures separation based primarily on molecular weight
Stacking and Resolving Gel System
PAGE uses a discontinuous buffer system:
Stacking Gel
- pH: 6.8
- Larger pore size
- Concentrates proteins into a thin band before entering the resolving gel
Resolving Gel
- pH: 8.0
- Smaller pore size
- Separates proteins based on size
This system improves resolution and produces sharp, distinct bands.