Poly Acrylamide Gel Electrophoresis
Electrophoretic techniques are laboratory techniques used to separate charged molecules in an electric field. The movement of a molecule is inversely proportional to its size and directly proportional to its charge.
Throughout electrophoresis, proteins move towards an oppositely charged electrode in an electric field. The rate of their movement in an electrophoretic system is governed by numerous elements such as temperature, pH, and buffer concentration in addition to intrinsic factors such as the size, charge, and shape of the proteins.
Polyacrylamide gel electrophoresis (PAGE) is a technique based on this concept and is utilized to separate proteins on the basis of their size. Polyacrylamide gel electrophoresis (PAGE) is a highly reliable and commonly utilized technique for the separation, identification, and characterization of proteins and protein mixes.
Principle of PAGE
In PAGE, an anionic cleaning agent called sodium dodecyl sulfate (SDS) is utilized to bind to proteins and give them a negative charge. Proteins are then separated electrophoretically according to their size using a gel matrix made of polyacrylamide in an electric field.
Polyacrylamide is produced as a result of the polymerization reaction between acrylamide and N, N’- methylene-bis-acrylamide (BIS) using a catalyst. The degree of polymerization or cross-linking can be managed by adjusting the concentration of acrylamide and BIS.
The more the cross-linking the harder the gel is. The firmness of the gel, in turn, regulates the friction experienced by macromolecules when they move through the gel during PAGE, therefore impacting the resolution of separation.
Loose gels (4-8% acrylamide) enable greater molecular weight molecules to move faster through the gel while hard gels (12-20% acrylamide) restrict the movement of big particles and selectively enable small ones to move through the gel.
Preparation of PAGE
It is prepared by polymerizing acrylamide monomers in the presence of methylene-bis-acrylamide to cross-link the monomers. Polyacrylamide gel structure is held together by covalent cross-links.
Polyacrylamide gels are harder than agarose gels. It is thermostable, transparent, strong, and reasonably chemically inert. Gels are uncharged and are prepared in a variety of pore sizes.
Equipment for PAGE
The equipment and products essential for carrying out PAGE consists of:
- An electrophoresis chamber and power supply.
- Glass plates (a brief and a top plate).
- Casting frame
- Casting stand
- Combs
Steps involved in PAGE
The gel of different pore sizes is cast into a column inside a vertical tube, typically with large pore gel at the top and little pore gel at the bottom. Microgram amount of the sample is positioned over the top of the gel column and covered by a buffer solution having such a pH so as to change sample elements into anions.
The foot of the gel column is made to dip in the same buffer in the bottom reservoir. Cathode and anode are kept above and below the column to impose an electric field through the column. Macromolecular anions move towards the anode down the gel column.
There is no external solvent space, all the migratory particles have to pass through the gel pores. The rate of migration depends on the charge-to-mass ratio. Various sample components get separated into discrete migratory bands along the gel column on the basis of electrophoretic movement and gel filtering results.
Slab PAGE
The Polyacrylamide gel is cast as a thin rectangle-shaped piece inside a plastic frame and this piece is put vertically on a buffer solution taken in a tank. Several samples dissolved in dense sucrose service or glycerol are put in separate wells cut into the upper edge of the piece and are covered by the exact same buffer solution.
Cathode and anode are above and below to produce electric field results. Different components move all at once down parallel lanes in the slab and get separated into bands.
Visualization
After the electrophoresis is complete, the molecules in the gel can be stained to make them visible. Ethidium bromide, silver, or Coomassie blue color may be utilized for this procedure. If the analyte molecules fluoresce under ultraviolet light, a photograph can be taken of the gel under ultraviolet lighting conditions. If the particles to be separated contain radioactivity included for visibility, an autoradiogram can be recorded of the gel.
Advantages:
- Gels are stable over a wide range of pH and temperatures.
- Gels of various pore sizes can be formed.
- Simple and separation speed is excellent relatively.