PAGE (Polyacrylamide gel electrophoresis)

What is PAGE ( Polyacrylamide gel electrophoresis ), Introduction, Principal , Process , flowchart ?

PAGE (Polyacrylamide gel electrophoresis) :-



PAGE  ( Polyacrylamide gel electrophoresis )
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Introduction :-


The term Electrophoresis describes the Movement or Migration of the Charge macromolecules or particles under the influence of the Charge, by the help of Electrophoresis we generally Separates and do analysis of the macromolecules like Nucleic acid, Protein, Amino acid Nucleotides etc. under the influence of an electric field these charge Particles will migrate or move either to the Cathode or to the Anode and this nature of movement generally depend on the nature of their electric charge.


Principle of PAGE :-


PAGE (Polyacrylamide gel electrophoresis) generally used in Biotechnology, Molecular biology, Genomics and Proteomics labs etc. to separate the Biological macromolecules according to their Electrophoretic mobility.
Electrophoretic mobility is function of length, conformation and charge of the molecule.

Process:-


The equipment required for electrophoresis consist basically of two items, a power pack and an electrophoresis unit. electrophoresis units are available in market for running either vertical and Horizontal systems. the power pack system supplies the direct current between the electrodes in the electrophoresis unit. all the electrophoresis process is carried out in an appropriate buffer, which is essential to maintain a constant state of ionization of the molecules being separated.

1) Sample preparation


Sample may be biologically derived for example From Eukaryotic or Prokaryotic cells, tissues, Purified proteins etc. in this case we generally broken down the solid tissues or cells by either mechanically by using Sonicator, Blender, Homogenizer etc. and then we go for Filtration and Centrifugation step if required, the sample of interest is then optionally mixed with chemical detergents usually SDS for Proteins. SDS is an anionic detergent that is use to denature the secondary, tertiary structures of protein. 
A dye that have the higher electrophoretic mobility then the analyte then added to the solution which is called as tracking dye that track the progress of the solution through the gel during the electrophoretic run.


2) Preparing acrylamide gels


The Electrophoresis gels consist of Acrylamide, Bisacrylamide , optionally Detergents and a PH adjusted buffer. ammonium persulfate and TEMED are added to initiate the polymerization and they are the sources of free radicals and stabilizer. the polymerization reaction results to creates the gel because for sample preparation we added bisacrylamide which can form cross links between two acrylamide molecules. the gels is polymerized between two glass plates in a gel caster, to create the sample wells we inserted a comb at the top. after polymerization we remove the comb and now we can perform our desired electrophoretic experiment.

3) Electrophoresis


Depending on the nature of the samples various types of buffers are used. the buffer that are used are may be same for both anode and cathode or may be not. when we applied the current across the gel the negatively charged particles moves towards the Anode. small molecules directly fit through the gel matrix but larger one have difficulties. the running time of the gel usually depends on the applied voltage across the gel. if we applied higher voltage across the gel the molecules migrate more quickly but these results are not accurate or less accurate as compare to lower voltage.


4) Further processing


The gels are stained that allow the visualization of the separated proteins or process further.
 distinct bands are appear of different proteins after staining further process example Western blotting.


Flow chart :-


Flowchart,  PAGE ( Polyacrylamide gel electrophoresis )
PAGE process flowchart











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