PAGE: Principle, Procedure & Component, Variations, Advantages & Disadvantages

• PAGE, which stands for Polyacrylamide Gel Electrophoresis, is a widely used laboratory technique for the separation of biomolecules, most commonly proteins and small nucleic acid fragments, based on their molecular weight.

Basic Principle:

• In PAGE, an electric field is applied across a gel matrix made up of polyacrylamide.

• As this happens, charged molecules (like proteins or DNA fragments) migrate through the gel.

• Their rate of migration is inversely proportional to their molecular weight – smaller molecules move faster through the matrix, while larger one's lag behind.

Here's the diagram illustrating the "Principle of PAGE (Polyacrylamide Gel Electrophoresis)

Procedure and Components of PAGE:

1. Gel Preparation:

• A solution of acrylamide and bis-acrylamide is mixed with a buffer, then polymerized using a catalyst (usually ammonium persulfate) and a stabilizer (like TEMED). This creates a mesh-like matrix when it solidifies.

2. Loading Samples:

• Once the gel has set, samples are loaded into wells created at the top of the gel. A ladder or molecular weight marker is often loaded alongside the samples to provide reference points for determining the size of the separated molecules.

3. Electrophoresis:

• The gel apparatus is filled with a running buffer, and an electric field is applied. Negatively charged molecules migrate towards the positive electrode (anode) at the bottom.

4. Visualization:

• After electrophoresis, proteins or nucleic acids in the gel can be visualized using various staining methods. Common stains include Coomassie Blue (for proteins) and ethidium bromide (for DNA).

Here's the diagram illustrating the "Variations of PAGE"

Variations of PAGE:

1. SDS-PAGE (Sodium Dodecyl Sulfate-PAGE):

• SDS is an anionic detergent that denatures proteins and gives them a negative charge proportional to their length. This means that, in SDS-PAGE, proteins are separated purely based on molecular weight rather than their native charge.

2. Native PAGE:

• Unlike SDS-PAGE, native PAGE doesn't use any denaturing agents. Proteins retain their native structures and charges, so they are separated based on both size and charge.

3. 2D-PAGE (Two-dimensional PAGE):

• In this advanced technique, proteins are separated based on their isoelectric points in the first dimension and then by their molecular weights in the second, providing a more comprehensive separation profile.

classification of variation of page

Advantages of PAGE:

1. Resolution:

• Polyacrylamide gels can resolve molecules of very similar sizes, providing high-resolution separations.

2. Versatility:

• PAGE can be used for both DNA and proteins, and by tweaking the conditions, various separation profiles can be achieved.

Here's the diagram illustrating the "Advantages of PAGE"

Disadvantages:

1. Toxicity:

• Acrylamide, a primary component of the gel, is neurotoxic and poses health risks.

2. Fragility:

• Polyacrylamide gels are more fragile compared to agarose gels, making handling more challenging.

• In summary, PAGE is a robust analytical technique in molecular biology and biochemistry, offering detailed insights into the molecular weight and composition of protein samples and small nucleic acid fragments.

Here's the diagram illustrating the "disadvantages of PAGE"