Electrophoresis and types of electrophoresis

• The term electrophoresis describes the moment of the molecule under the influence of the electric field.
• Many important biological molecules, such as amino acids, peptides, proteins, nucleotides, and nucleic acids, possess ionizable groups and, therefore, at any given pH, exist in solution as electrically charged species either as cations (+) or anions (-). 
• Under the influence of an electric field, these charged particles will migrate either to the cathode or to the anode, depending on the nature of their net charge.
• Electrophoretic mobility (μ) directly proportional to the applied electric field (E).
• μ ∝ E
• μ ∝ q (charge on the biomolecule)
• μ ∝ 1/f (friction force on the biomolecule)
• friction force of biomolecule is depended upon the shape and size of the biomolecule.
• Heating of the electrophoretic medium has the following effects:
• (i) An increased rate of diffusion of sample and buffer ions leading to the broadening of the separated samples.
• (ii) The formation of convection currents, which leads to the mixing of separated samples.
• (iii) Thermal instability of samples that are rather sensitive to heat. This may include the denaturation of proteins or loss of activity of enzymes.
• (iv) A decrease of buffer viscosity, and hence a reduction in the resistance of the medium.

Agarose Gels Electrophoresis (AGE): 

"Agarose gels are used in techniques such as immunoelectrophoresis or flatbed isoelectric focusing, where proteins are required to move unhindered by their native charge in the gel matrix"

• Agarose gels are used both for protein electrophoresis and for nucleic acids. 
• 1 percent agarose gel pore sizes are large relative to protein sizes. 
• Agarose gels are used in techniques such as immunoelectrophoresis or flatbed isoelectric focusing, where proteins are required to move unhindered by their native load in the gel matrix.
• Such large porous gels are used to separate much larger molecules, such as DNA or RNA because the pore sizes of the gel is still large enough to allow DNA or RNA molecules to pass through the gel.
• The size of the pores and the size of the molecules are more comparable and the frictional effects continue to play a role in the separation of these molecules.
• The availability of low melting agarose temperatures (62 to 65⁰C) is another benefit of the use of agarose.
• For example, these gels can be heated to 65⁰ C and reliquified.
• DNA samples isolated in a gel can be removed from the solvent, returned to the solution, and recovered.
• The gel rod method is often used for early due to the low elasticity of agarose gels and the consequent difficulty in extracting them from short tubes. 
• Mid-size hot gels used. 
• Invariably, horizontal slab gels are used for isoelectric focusing or immunoelectrophoresis in agarose. 
• Horizontal gels are commonly used for DNA and RNA gels, with some personnel using vertical systems.

Source: ScienceDirect

Keyword: nucleic acid; immunoelectrophoresis; a large molecule; electrophoresis; agarose gel

Polyacracylamine gel

Electrophoresis of acrylamide gels is also referred to as PAGE, which is an abbreviation for electrophoresis of the polyacrylamide gel. In the presence of smaller quantities of N, N -methyl, cross linked polyacrylamide gels are formed from the polymerization of the acrylamide monomer. Enebisacrylamide (commonly referred to as 'bis'-acrylamide).

“Acrylamide monomer is polymerized head-to-tail into long chains and sometimes a bis-acrylamide molecule is inserted into it. Chain, thus adding a second chain expansion site”

Source: Microbes notes

• Acrylamide monomer is polymerized head-to-tail in long chains and sometimes a bis-acrylamide molecule is inserted into the expanding chain, adding a second chain expansion site. 
• Acrylamide polymerization is an example of free radical catalysis induced by the addition of ammonium persulphate and base N, N, N, N', N'-tetramethylene diamine (TEMED).
• TEMED catalyzes the decomposition of the persulphate ion to produce a free radical: S2O8-+ e-=SO42-+ SO4-. 
• Forming a single bond by sharing its unpaired electron with one of the outer shells of the monomer molecule. This produces a new free radical molecule, R-M, which is reactive and attacks another monomer molecule. 
• Long chains of acrylamide are thus built up, interlinked by the introduction of an occasional bis-acrylamide molecule into the growing chain. 
• Oxygen eliminates free radicals and all gel solutions are typically degassed prior to use. 
• Photopolymerization is an alternative process for the polymerization of acrylamide gels.
• Riboflavin replaces ammonium persulphate and TEMED, and when the gel is poured, it is placed in front of bright light for 2 to 3 hours. 
• The photodecomposition of riboflavin produces a free radical that initiates polymerization.
• Acrylamide gels are specified in terms of the total percentage of acrylamide present and the pore size of the gel may vary by adjusting the concentrations of both acrylamide and bis-acrylamide.
• Low-percentage gels (e.g. 4 percent) have large pore sizes and are used, for example, in protein electrophoresis where free movement of proteins by electrophoresis is required without any visible frictional effect, e.g. in isoelectric flatbed focusing or in the SDS-polyacrylamide gel stacking system. 
• Acrylamide gels are used to isolate a small amount of DNA.
• Gels between 10% and 20% of acrylamide are used in techniques such as SDS-Gel electrophoresis, in which smaller pore sizes produce a sieving effect that leads to the separation of proteins by their thickness. 
• The proteins were initially isolated by polyacrylamide gels which were polymerized in glass tubes, approximately 7 mm in diameter, and approximately 10 cm in length.
• The subsequent installation of vertical gel slabs allowed up to 20 samples to run on a single run under similar conditions. 
• Vertical slabs are commonly used for both protein analysis and DNA fragment separation during DNA sequence analysis.

Keyword: TEMED; acrylamide monomer; polyacrylamide gel electrophoresis; single bond; free radical; methylene group; long-chain; unpaired electron

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