Methods - Method Used to Amino Acid Hydrolysis and...

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Method Amino Acid Hydrolysis and Chromotography Sanger Method Carboxypeptidase Edman Used to Identify amino acids present in vitro Identify the primary structure of a protein Identify primary struc- ture of a protein Identify primary structure of a protein Procedure Load column so all amino acids stick. Elute each amino acid so they come off one at a time. Use a non-polar matrix of SO3- beads and make act- ive by raising pH above 1.5. pH also should be less than 3 so no charge on aspartic acid. Load using 2.2M buffer. Get pure protein and seperate subunits using mercaptoethanol. Do amino acid analysis (chromo- tography) to find which are present. Use Sanger’s reagant to dye n-terminus yellow. Break off n-labelled yellow by using A + B Carboxypeptidase (an endopro- tease). Generate overlapping se- quences. A c-terminus analysis. Use A + B carboxypeptidase to cut off “C”, use a free amino acid (no color) and run TLC. Use a new chemical: phenylisothiocyanate (PITC). Uses no water, use anhydrous triflouro- acetic acid to provide pro- ton. PITC ill be reporter and in an elimination reac- tion @50 O C, will produce PTI-amino acid Assay Collect 2ml in test tube, put .1 ml, ninhydrin, and head. 19/20 -> Yellow. Proline -> Purple. Get off column using an elu- tion buffer. Can elute by chan- ging pH or raising salt concen- tration. Use 3 buffers Use thin layer chromatography. Put yellow aliquot on TLC plate.
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This note was uploaded on 04/23/2008 for the course BIS 102 taught by Professor Hilt during the Winter '08 term at UC Davis.

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Methods - Method Used to Amino Acid Hydrolysis and...

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