IndietroPrinciples of Electrophoresis in General Biology
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Electrophoresis is a bioanalytical technique used to separate charged macromolecules based on their mobility in an electric field, influenced by charge, molecular weight, and shape.
Positively charged species migrate toward the cathode, and negatively charged species migrate toward the anode.
Electric field strength, E, is given by \(E=\frac{\text{applied voltage}}{\text{length of support medium}}\).
Because the support medium has low conductivity, acting like a resistor between capacitors at electrode interfaces, causing a linear potential drop and preventing electrolysis.
The electric force qE equals the viscous drag fv, where q is charge, E is electric field, f is frictional coefficient, and v is velocity.
Electrophoretic mobility, m, is the velocity per unit electric field strength, given by \(m=\frac{v}{E}=\frac{q}{f}\).
Mobility increases with charge q, decreases with frictional coefficient f (related to size and shape), and is zero for uncharged particles.
Moving-boundary electrophoresis, zone electrophoresis, and steady-state electrophoresis (e.g., isoelectric focusing).
Analyte solution with high-density nonionizing component migrates in a U-shaped cell, with boundaries moving toward electrodes; used for precise mobility measurements.
Separation of analyte mixture into discrete bands or zones on a support medium; widely used for molecular weight and purity analysis.
Zones reach constant positions and widths over time; example is isoelectric focusing where molecules focus at their isoelectric points.
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.
Separation depends on charge and size; starch gels have sieving and ion-exchange properties and exhibit electroosmosis.
Separation technique using polyacrylamide gels with controllable pore size; widely used for proteins and small nucleic acids.
A PAGE method using a stacking gel with lower pH and ionic strength to concentrate samples 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 sodium dodecyl sulfate denatures proteins and imparts uniform charge, allowing molecular weight determination independent of native charge.
Separation of large nucleic acids and proteins due to larger pore size; gels are temperature sensitive and used in DNA analysis.
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; proteins migrate toward cathode if pH < pI and toward anode if pH > pI.
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 techniques transferring molecules to membranes.
Transfer of DNA from agarose gels to membranes for hybridization-based detection using labeled DNA probes.
Transfer of RNA from gels to membranes for detection; requires special denaturation and RNase-free conditions.
Transfer of proteins from gels to membranes for immunodetection using antibodies.
By hybridization through complementary base pairing between labeled probes and target nucleic acids on membranes.