Experiment 2

Experiment 2 - CHEM 241L: The Determination of the...

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CHEM 241L: The Determination of the Isoelectric Point of a Protein 52 2 Experiment 2 The Determination of the Isoelectric Point of the Protein Bovine Serum Albumin Read section 10-6 (Chapter 10) and the section entitled “Electrophoresis” in section 26-5 (Chapter 26) of Harris, 7 th ed. BACKGROUND & THEORY The electrophoresis experiment is the practical application of the idea that a charged molecule will move (migrate) in an electric field. Electrophoresis is a separation technique that is widely used by biologists, chemists, and biochemists The experiment you will perform today utilizes a slab electrophoresis format. Although it will not be discussed here, you should be aware that electrophoresis is also commonly performed in capillaries (capillary electrophoresis). Slab electrophoretic separations are typically carried out on a thin flat layer or slab of some porous semisolid gel or membrane contained in an aqueous buffer solution. 1 Paper electrophoretic strips made from cellulose polyacetate will be used in this experiment. The ends of the paper strips are immersed in separate reservoirs containing aqueous buffer and an electrode. The aqueous buffer wets the entire paper strip and thus electrically connects the two buffer reservoirs. A power supply provides a voltage (V) between the two electrodes and an electric field (voltage gradient) is established along the liquid strips. The electric field (E) is determined by dividing the applied voltage by the distance (d) between the two electrodes as illustrated in Figure 1. The sample is placed in a spot or line across the center of the strip (the sample origin ), as illustrated in Figure 1. When a voltage is applied an electric field is established and the charged sample will migrate in the electric field. After some predetermined time the voltage is turned off and the sample is detected by chemical means. Reading Assignment
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CHEM 241L: The Determination of the Isoelectric Point of a Protein 53 T h e direction of migration is determined by the net sign (positive or negative) of the molecules charge. Positively charged molecules will migrated towards the cathode (the negative electrode), negatively charged molecules will migrate towards the anode (the positive electrode), and molecules with a net zero charge will not migrate. The direction of migration is simply governed by the electrostatic attraction between the charged molecules and the charged electrodes. The rate at which the charged molecule migrates (u ep ) is governed by the electrophoretic mobility ( μ ep ) of the sample molecule and by the magnitude of the electric field (E), as illustrated in Expression (1). 2 E u ep ep μ = (1) d V E = Figure 1. Schematic of a basic electrophoretic setup and illustration of how the electric field _ Paper Strip Buffer Reservoir Buffer Reservoir Cathode (- electrode) Anode (+ electrode) Barrier Between Buffer Reservoirs d Power Supply (V) Sample Origin +
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CHEM 241L: The Determination of the Isoelectric Point of a Protein 54
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This note was uploaded on 02/21/2010 for the course CHEM 241 taught by Professor Tiani during the Spring '08 term at UNC.

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Experiment 2 - CHEM 241L: The Determination of the...

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