sequencelabs02

sequencelabs02 - Chemistry 365 Biochemistry Individual Lab...

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ser glu ala phe lys met leu glu Ion Exchange Chromatography, Sulfonated Polystyrene Resin Elution Profile of Amino Acids Elution Volume S E A F K M L E pH 3.25 0.2 M citrate pH 4.25 0.2 M citrate pH 5.28 0.35 M citrate Chemistry 365 Biochemistry Individual Lab Unit #7 Sequence Determination Techniques for Proteins and Nucleic Acids References: Lehninger, A.L.; Nelson, D.L.; Cox, M.M. Principles of Biochemistry , 2nd ed.; Worth Publishers: New York, 1993. Berg, J.; Tymoczko, J.; Stryer. L. Biochemistry , 5 th ed.; W. H. Freeman: New York, 2002. Introduction: I. Determining Protein Amino Acid Sequence Once the protein of interest has been extracted and purified, and its molar mass determined, the next step is to completely hydrolyze the protein (6 N HCl at 110 o C for 24 hours) and determine its amino acid composition. Amino acids in the hydrolysate are separated and identified by ion exchange chromatography. In most cases, the next step is to identify the first amino acid in the sequence, or in other words, the protein's amino terminal. The amino terminal is typically reacted with a labelling reagent, such as dabsyl chloride, that forms a bond stable to hydrolysis. The peptide is hydrolyzed and the labeled amino acid identified. Peptides up to 50 residues long can be sequenced by a cyclic procedure where the amino terminal is labelled, cleaved, identified and the process repeated on the shortened chain. This procedure, the Edman degradation method, has
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sequencelabs02 - Chemistry 365 Biochemistry Individual Lab...

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