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Wednesday, May 26, 2021

Gel Electrophoresis of Protein - CSIR/ DBT/ ICMR (Life Sciences)

Gel Electrophoresis of  Protein

Gel Electrophoresis:

Features:

  • Gel matrix is the stationary phase and liquid (buffer with various ions) is the mobile phase.

  • Gel matrix is made up of some polymers viz. polyacrylamide polymer or agarose polymer.

  • The gel is a three-dimensional molecular network and this molecular network has various pores through which molecules can pass. Hence, it acts as a mobile sieve.


  • The distribution of the pore size in a gel determines the size range of the ions that can be separated.

Theory of Gel Electrophoresis:

A molecule with net charge 'q' moves in an electric field with velocity 'v'.

v = E*q/f

Where, 

  • 'E' is electric field in volts/cm. 
  • 'f' is a frictional coefficient, which depends upon the shape and mass of the molecules.

Experiments are typically done at a constant voltage. Then the movement of molecules depends only on q/f.

For molecules of similar shapes, such as DNA or SDS-bound proteins, the movement depends only on the size of the molecules. Small molecules move faster than large molecules.


Methods of gel electrophoresis:

Polyacrylamide Gel Electrophoresis (PAGE):

  • Used to separate large biomolecules such as proteins and DNA.
  • It typically runs under two conditions: denaturing and native.
  • High-resolution power for biomolecules up to 1000 kDa.
  • Gel matrix has good physical stability, thus, easy to handle.
  • Sample separation is due to both molecular sieving and electrophoretic mobility.
  • No void volume in the matrix. An only continuous network of pores.
  • Small molecules move faster than larger molecules.
  • The pore size of the gel matrix is controlled by the concentration of acrylamide and N, N'-methylenebisacrylamide.
  • Caution: Monomer acrylamide is a neurotoxin and is also suspected to be a carcinogen.
acrylamide (4 to 20%)
Fig: acrylamide (4 to 20%)


N, N'-methylenebisacrylamide (1 to 5% of acrylamide)
Fig: N, N'-methylenebisacrylamide (1 to 5% of acrylamide)

Polyacrylamide Matrix
Fig: Polyacrylamide Matrix



Advantages of Polyacrylamide Gel:

  • It does not interact with protein or nucleic acid.
  • It is chemically stable, hydrophilic, free of ions.
  • It does not interfere with common staining reactions.


SDS-PAGE denaturing condition:

Discontinuous Gel Electrophoresis:

  • Two gel layers: stacking gel (upper) and resolving gel (lower).
  • These are prepared with buffers with different ionic strengths and pH.
  • Stacking gel concentrates all protein molecules into a tight band before entering the resolving gel.
  • Sodium dodecyl sulfate (SDS) is a detergent.
  • SDS binds hydrophobic regions of the denatured protein, resulting in a constant charge/mass ratio and uniform shape. Proteins are now separated by size.
  • The majority of the current is carried by the buffer ions i.e.tris, glycine, and chloride.
  • Protein ions have a negligible contribution to the current.
  • At low buffer concentration, proteins will migrate fast, resulting in smeared bands.
  • At high buffer concentration, proteins will move very slowly.
  • Visualizing the protein bands with Bromophenol blue (negatively charged and high electrophoretic mobility, runs fastest), Coomassie Brilliant Blue R-250, and Coomassie Brilliant Blue G-250.

Functions of Stacking gel:

  • The stacking gel is polymerized from a dilute acrylamide solution. The large pore size does not provide a molecular sieving effect.
  • Stacking gel ensures that all of the proteins arrive at the running gel at the same time so proteins of the same molecular weight will migrate as tight bands.

Functions of Resolving gel:

  • The resolving gel is to separate the protein based on their molecular weight.


Processes:

  • Protein purified sample incubated with Sodium Dodecyl Sulfate (SDS) to provide a uniform negative charge.
  • Sample from different animals loaded into different "wells" of an electrophoresis chamber.
  • Acrylamide gel, containing microscopic pores, separates the cathode/anode.
  • The first "well" contain loading control. A mixture of multiple proteins of known molecular mass added as a reference to experimental wells. Tracking dye added to loading control to visualize protein migration.
  • When electrical current is run through the chamber, negatively charged proteins migrate towards a positive anode.
  • Smaller proteins "slip" through resolving gel more quickly than larger bulky proteins.
  • Visualization of protein by western blotting.

SDS- Polyacrylamide Gel Electrophoresis

Fig: SDS- Polyacrylamide Gel Electrophoresis





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