Proteins and Genes Report

Proteins and Genes Report - of Proteins by...

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of Proteins by Ion Exchange Chromatography Separation of Proteins by Ion Exchange Chromatography: Introduction: Each protein is unique and distinct in their properties. These properties include size, shape, function and charge. Ion exchange chromatography uses the unique characteristics of the proteins to separate them. Column ion exchange chromatography involves a thick slurry matrix which can be either anion or cation exchangers. Because of their unique features, the proteins react to the matrix differently. The difference in charge means that the column which is made up of small beads that sustain and conduct positive or negative charges that delay proteins of the opposite charge. The pH determines the ionization state of the molecule. This means that the protein’s attraction to another charged molecule becomes either higher or lower at different a pH due to the free flowing salt ions surrounding the solution. Aim: To understand the principle of ion exchange chromatography To observe the way that a protein’s charge varies to pH To obtain some information about the pI of each protein separated Method and Materials: Students will work in pairs . Each student pair will be provided with the following reagents: DEAE-cellulose slurry equilibrated in 10 mM phosphate buffer, pH 7.3 Buffer solutions 10 mM phosphate buffer (pH 7.3) 30 mM phosphate buffer (pH 6) Sodium phosphate (30 mM) – citric acid (pH 4) Protein mixture in 10 mM phosphate buffer pH 7.3: Horse cytochrome C (1.0%, w/v) Bacillus subtilis α -amylase (0.5%, w/v) Bovine serum albumin (1.0%, w/v) 1% starch solution Iodine solution (0.2% I 2 in 2% KI diluted 10-fold with H 2 O) Preparation of DEAE Cellulose DEAE-cellulose supplied in a pre-swollen form (DE52) has been equilibrated for you already by the following procedure: 250 ml of 10 mM phosphate buffer, pH 7.3, was added to 50 g of DE52 and the pH of the mixture adjusted to pH 7.0 by adding 1M HCl. Jonathon Nguyen | 16323012
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The slurry was allowed to settle for 30 min, the supernatant and any "fines" poured off and the slurry was resuspended in 250 ml of 10 mM phosphate buffer. The washing cycle was repeated twice more. The slurry was filtered through Whatman No.1 filter paper on a Buchner funnel and the cellulose "cake" resuspended in 50 ml of 10 mM phosphate buffer. The ion exchanger was then ready to use. Preparation of the DEAE-Cellulose column
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This note was uploaded on 06/15/2010 for the course AP 31239 taught by Professor Meher during the Spring '10 term at Acton School of Business.

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Proteins and Genes Report - of Proteins by...

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