Topic%2006a%20_%20Electrophoresis%20_%20F10

Topic%2006a%20_%20Electrophoresis%20_%20F10 -...

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Unformatted text preview: Polyacrylamide Gel Electrophoresis of Proteins • Electrophoresis: migration of charged molecules in a solution or gel matrix under the influence of and electric field • Protein X with a net negative charge moves towards the anode X------- +- anode cathode moves towards anode How fast does protein X move towards the anode? • In a vacuum: remember physics………… Protein X continues to accelerate in an electric field until it hits something (mass spectrometers); and the acceleration rate is dependent on the charge/mass ratio. F electric field = ma = qE force = mass x acceleration = net charge x electric field a = (q/m)·E • In solution through a matrix ( ie a gel), a charged molecule will initially accelerate but eventually reach a terminal velocity due to the frictional drag of colliding molecules. F electric field = ν f terminal velocity frictional coefficient = qE ν = (q/f)·E Particle velocity = (q/ f )·E = v f = frictional coefficient = 6 πη r f is a complex variable dependent on the viscosity of the medium, and the mass and shape of the particle viscosity of particle radius the medium for a sphere mobility ( μ ) = E = q f Formal Definition of Electrophoretic Mobility: Units of mobility: mobility ( μ ) = E = cm/sec volts/cm = cm 2 volts • sec ν ν Practical purposes: if proteins are spheres of similar densities (“close packing”), and in a system with a defined matrix and buffer solution, then mobility ( ie terminal velocity) is easiest to view as: ν ~ (q/m)·E 2 H 2 O + 2 e − → H 2 + 2 OH − 2 H 2 O → 4 H + + O 2 + 4 e − cathode − + anode power supply electrons come from the power supply electrons return to the power supply sample (net negatively charged proteins) buffered medium to conduct current and resist pH changes Electrophoresis Media for Electrophoresis...
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Topic%2006a%20_%20Electrophoresis%20_%20F10 -...

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