IndietroPrinciples of Electrophoresis in Analytical Chemistry
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Electrophoresis is a bioanalytical technique used to separate charged macromolecules based on their mobility under an electric field, depending on charge, molecular weight, and shape.
Positively charged species migrate toward the cathode, and negatively charged species migrate toward the anode in the electric field.
Electric field strength, \(E\), is given by \(E=\frac{\text{applied voltage}}{\text{length of support medium}}\).
Because the support medium has low conductivity, creating a resistor in the circuit that causes a linear voltage drop and prevents high field strengths at electrode interfaces that cause electrolysis.
Electrophoretic mobility, \(m\), is the velocity per unit electric field strength, given by \(m=\frac{v}{E}=\frac{q}{f}\), where q is charge and f is frictional coefficient.
Mobility increases with charge (q), decreases with frictional coefficient (f), and is zero for uncharged particles; also affected by ionic shielding and experimental conditions.
Performed in a U-shaped cell with a dense analyte solution; boundaries between analyte and buffer move under an electric field, monitored by refractive index detectors.
Separation of analyte components into discrete bands or zones on a support medium; widely used for biomolecule analysis and purity determination.
Zones reach constant positions and widths over time; example is isoelectric focusing where molecules migrate to their isoelectric point.
Paper, starch gels, polyacrylamide gels, agarose gels, and composite polyacrylamide-agarose gels.
Less adsorption and tailing, faster separations, lower background staining, transparency, and easier elution of components.
Made by heating potato starch paste; semisolid gel with limited pore size control, negatively charged side chains causing ion-exchange and electroosmosis effects.
Separation technique using polyacrylamide gels with controllable pore size; superior to starch gels for proteins and small nucleic acids.
By reaction of acrylamide with N,N'-methylenebis(acrylamide) using initiators like ammonium persulfate and catalyst TEMED.
A PAGE method using a stacking gel with lower pH and ionic strength to concentrate proteins into narrow bands for improved resolution.
By running gels with constant crosslinker %C and varying total monomer %T, calculating Rf values, and constructing Ferguson plots.
PAGE with SDS detergent that denatures proteins and imparts uniform charge-to-mass ratio, allowing molecular weight determination independent of charge.
Separation of large nucleic acids and proteins due to larger pore size; gels are temperature sensitive and used in vertical or horizontal modes.
Temperature influences convection, diffusion, band distortion, evaporation, and gel viscosity; cooling is essential for resolution.
Protein net charge depends on pH relative to isoelectric point (pI); below pI proteins are positive, above pI negative, affecting migration direction.
Higher ionic strength shields charges, reducing mobility and affecting separation quality; low ionic strength buffers are preferred.
To improve resolution of slow-moving species by increasing field strength near the origin using wedge-shaped gels or ionic strength gradients.
By staining with dyes (e.g., Coomassie Blue, silver stain) or activity stains, or by blotting onto membranes for further analysis.
Transfer of denatured DNA from agarose gels to nitrocellulose or nylon membranes for hybridization and detection.
Transfer of RNA from gels to membranes using denaturation methods that avoid RNA hydrolysis, followed by detection.
Transfer of proteins from gels to membranes, often by electrophoretic transfer, for immunodetection or other analyses.
By hybridization through complementary base pairing of labeled oligonucleotide probes to target DNA on membranes.