BME 210 Lecture 13 Electrophoresis and Blotting

BME 210 Lecture 13 Electrophoresis and Blotting - 13....

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13. Electrophoresis and Blotting
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Principle of Electrophoresis Electrophoresis - movement of electrically charged particles in a liquid environment due to electric field • Forces and particle movement: F e = qE F f = vf ma = F e – F f F e – electric force E – field strength q – particle charge F f - friction force; f - friction coefficient v – particle velocity; a – acceleration; m - mass • Due to F e , v increases until F f becomes equal to F e a = 0 • Steady-state velocity v: qE = vf v = Eq/f E v q F e F f + -
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Separation factors v = Eq/f • Particles moving at different velocities become spatially separated • For the same E, particle separation is determined by the q/f ratio: Particle charge Friction coefficient - function of particle shape and size, and medium viscosity
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Gel electrophoresis • EP is run in a gel slab: • The gel is solid to prevent convection • It contains water and electrolytes • There are pores allowing molecule to pass • Molecular mixtures are loaded on top of the gel slab • Electric field ≈ 5-20 V/cm • Negatively charged molecules move towards the anode; positively charged - towards the cathode • Lateral diffusion of molecules is negligible – they move in lanes • Gel EP is used for separation of DNA, RNA and proteins
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EP of double-stranded nucleic acids DNA and RNA are negatively charged at any pH (because of their phosphate groups) • Repulsion between the charged groups straightens out double-stranded DNA fragments ; not very long dsDNA behave as rods • Small molecules have much lower friction than large molecules Small molecules migrate faster despite their smaller charge (friction wins over charge) • The distance of travel, x , is approximately inversely proportional to the logarithm of the molecule length, which is proportional to molecular weight, MW: x ~ 1/log(MW) Separation of double-stranded DNA is according to their MW DNA
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EP of single-stranded nucleic acids Single-stranded DNA and RNA may fold into complex structures their migration rate is not a simple function of MW (shape is important as well) • Before EP, they are denatured and straightened out chemically (e.g., by high pH) and they become more rode-shaped • Now, similar to the double-stranded DNA, EP separation of single-stranded DNA/RNA is determined by their MW t-RNA
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DNA visualization and MW measurement DNA is visualized by staining with a fluorescent dye an absorption dye, e.g., ethidium bromide (generic DNA stain; observation in UV light) MW measurement : by comparison with electrophoretic patterns of mixtures of DNA fragments of known length ( DNA ladders; commercially available ) DNA ladders DNA samples bp bp - base pairs
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EP Gels 1. Polyacrylamide gel: small pores used for separating small nucleic acids ( MW <≈ 1000 bp ) high sensitivity ( ≈one nucleotide ) 2. Agarose gel (seaweed extract): large pores used for separating large DNAs ( MW <≈ 50,000 bp
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BME 210 Lecture 13 Electrophoresis and Blotting - 13....

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