Polyacrylamide Gel Electrophoresis (PAGE)

Polyacrylamide Gel Electrophoresis (PAGE) separates proteins by size and charge with high resolution in a polyacrylamide gel for analysis.

Polyacrylamide Gel Electrophoresis (PAGE)

• 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

1. 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.

2. Loading Samples:

   • Samples are loaded into wells created in the gel, along with a molecular weight marker (ladder) for size reference.

3. 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.

4. 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

1. High Resolution: Capable of separating molecules with very similar sizes.
2. Versatility: Suitable for proteins and nucleic acids; conditions can be tailored for various applications.

Disadvantages

1. Toxicity: Acrylamide is a neurotoxic substance requiring careful handling.
2. Fragility: Polyacrylamide gels are delicate and more difficult to handle compared to agarose gels.

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