Wednesday, 21 April 2021

NATIVE PAGE - Principle



  PAGE stands for Poly Acrylamide Gel Electrophoresis. This technique is mainly used to analyse proteins and small fragments of nucleic acids. Here we will focus on separation of proteins using PAGE. As the name indicates Poly Acrylamide gel used in this method

why we use polyacrylamide gel instead of agarose gel ?
The answer to this question is hidden in the average pore size of gel matrix in each case. Agarose gel has larger pores as compared to polyacrylamide gel, which has smaller pores.

 
   Therefore agarose gel is suitable for electrophoresis of large molecules, such as DNA and poly acrylamide gel is suitable for the electrophoresis of smaller molecules such as proteins.

Various factors on the basis of which proteins get separated
  In polyacrylamide gel electrophoresis separation of proteins depend on their
- charge density (charge to mass ratio)
- size (or Molecular weight) and shape.

Charge density (charge to mass ratio) of the protein molecule
  Proteins are composed of amino acids. Each of these amino acids carries charge either positive or negative. Also some amino acids may have no charge.
   Thus because of these individual charges on the amino acid residues, the protein molecule will carry an overall or net charge.
   The Net charge carried by a protein molecule depends on pH of its surroundings. So the net charge on a protein molecule can be positive negative or neutral at a particular pH in gel electrophoresis.
   pH of the buffer is set such that all the protein molecules at that pH will carry a negative net charge. Being negatively charged these protein molecules will migrate to the positive electrode or anode.

  Suppose there are two protein molecules in a given sample.
The first one has higher charge density and the second one has lower charge density as compared to the first.
The protein molecule with greater charge to mass ratio or charge density migrates faster in the gel.

Size and Shape of Protein molecules
   Proteins are of different sizes and shapes. Size is due to the number of amino acid residues in the protein molecule. This also means larger the size higher will be its molecular weight.
   The shapes of protein molecules they exist in various forms, Such as globular, elongated etc. The shape of protein depends on their ability to form primary, secondary, tertiary and quaternary structures.
   The extent of cross-linking and the average pore size of the gel affects the migration of proteins of various shapes and sizes.
Size -
   As larger the protein molecule, slower will be its migration. This is because larger protein molecules become entangled in the molecular sieve formed by the gel.
Shape -
  Compact globular proteins migrate faster than elongated proteins of comparable molecular weights.

So in page migration of proteins depend on the combination of multiple factors these are
- charge density
- size and shape of the protein molecules.
Here, The protein molecules remain in their Native form or in other words they remain intact. This type of page in which intact proteins in a sample are separated is known as Native page.
  This method is preferred when our requirement is to detect a particular protein on the basis of its biological activity. For example
• Separation and detection of enzymes
  Protein electrophoresis using native page has also some limitations. For example native page is not suitable for determination of molecular weights of proteins. 

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