PAGE is a widely used technique for separating biomolecules, especially proteins and small nucleic acid fragments, based on their molecular weight.
Basic Principle
Charged molecules migrate through a polyacrylamide gel matrix under an electric field.
Smaller molecules move faster through the gel, while larger ones move more slowly, as migration is inversely proportional to molecular weight.
Procedure and Components
Gel Preparation:
A solution of acrylamide and bis-acrylamide is mixed with a buffer.
Polymerization is induced using ammonium persulfate (catalyst) and TEMED (stabilizer), forming a mesh-like gel matrix.
Loading Samples:
Samples are loaded into wells created in the gel, along with a molecular weight marker (ladder) for size reference.
Electrophoresis:
The gel is placed in an electrophoresis apparatus filled with running buffer.
An electric field is applied, causing negatively charged molecules to migrate toward the anode.
Visualization:
After separation, molecules are visualized using stains:
Coomassie Blue for proteins.
Ethidium Bromide for DNA.
Variations of PAGE
1) SDS-PAGE:
Uses Sodium Dodecyl Sulfate (SDS) to denature proteins and give them a uniform negative charge.
Separation is purely based on molecular weight.
2) Native PAGE:
No denaturing agents are used, so proteins retain their native structure and charge.
Separation is based on both size and charge.
3) 2D-PAGE:
Combines isoelectric focusing (separation by isoelectric point) and SDS-PAGE (separation by molecular weight) for a detailed separation profile.
Advantages
High Resolution: Capable of separating molecules with very similar sizes.
Versatility: Suitable for proteins and nucleic acids; conditions can be tailored for various applications.
Disadvantages of Polyacrylamide Gel Electrophoresis
Toxicity: Acrylamide is a neurotoxic substance requiring careful handling.
Fragility: Polyacrylamide gels are delicate and more difficult to handle compared to agarose gels.