exp4 - Anusha Vadlamudi Partner: Christen Burns TA: Lindsay...

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Anusha Vadlamudi Partner: Christen Burns TA: Lindsay Aldridge Chem 241 Lab; Section 401; Room 300 Experiment 4: The Determination of the Isoelectric Point of the Protein Bovine Serum Albumin (5/21/08)
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Honor Code Pledge: I pledge that no unauthorized assistance has been given or received in the completion of the work presented in this report. Introduction The objective of this lab is to determine the isoelectric point of bovine serum albumin (BSA) using electrophoresis. Electrophoresis is a separation technique that employs an electric field and a buffer solution to separate species based on their charge and size. This experiment will use a slab electrophoresis format, utilizing a thin strip of electrophoresis paper made from cellulose polyacetate. The paper’s porous and semi- solid material allows the species to migrate. The paper is immersed in the aqueous buffer, and hence electrically connects the two buffer reservoirs like a salt bridge. After preparing the strips, the species of interest shaped as a line, is applied in the middle of the strip; this is called the sample origin. The ends of the strip of paper are immersed in the buffer solution, with one end in the cathode reservoir and one end in the anode reservoir. When a power supply provides a voltage, an electric field is created along the liquid strips. The formula for the electric field is: d V E = Where E stands for electric field, V for Voltage, and d for distance between two electrodes. The direction of migration is determined by the sign of the charge of the molecule. The migration direction is based on the principle that opposites attract. Negatively charged molecules will migrate toward the cathode (-), positively changed molecules will migrate toward the anode (+) and neutral molecules will remain at the sample origin. The rate of migration depends on the electric field (E) and electrophilic mobility ( μ ep ). The μ ep depends on molecule’s total charge (q) and the frictional coefficient (f):
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f q ep = μ The value of the frictional coefficient depends on hydrodynamic radius, shape, and viscosity. E f q E u ep ep = = As can be seen from the above equation, the charge is directly proportional to the rate of migration and therefore the larger the charger, the further it will migrate, and vice versa. Also, the lower the frictional forces, such as size and weight, the further it will travel. As in the larger the molecule and the higher the molecular weight, the shorter the migration distance. In conclusion, the charge and size influence the distance and the direction of a species’ migration. Isoelectric point is the pH at which the average charge of a polyprotic acid is zero. For amino acids, it can be found using the following formula: 2 2 1 a a pK pK pI + = Where 1 a pK is the pH at which the amino acid loses its first hydrogen and 2 a pK is the pH at which the amino acid loses its second hydrogen. Since BSA is a protein made up of several amino acids, its not possible to calculate the pI using the above formula. However, electrophoresis can be used to determine the isoelectric point of BSA.
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This note was uploaded on 02/23/2010 for the course CHEM 241L taught by Professor Domenictiani during the Spring '08 term at UNC.

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exp4 - Anusha Vadlamudi Partner: Christen Burns TA: Lindsay...

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