created: last updated: 26th June 1998,© 1998 ABRF



ABRF '96: Biomolecular Techniques

Amino Acid Analysis Tutorial

Improving the Art and Practice of Amino Acid Analysis

Karen A West, Jeffrey D Hulmes and John W Crabb

Protein Chemistry Facility
W Alton Jones Cell Science Center, Lake Placid, NY 12946
http://www.northnet.org/ajpchem

Sunday March 31, 1996
10:50 AM-12:20 PM
California Room
Holiday Inn Golden Gateway
San Francisco, CA


Practical approaches to improving bench level amino acid analysis efforts will be the focus of this tutorial. Amino acid analysis is one of the best quantitative methods in protein chemistry and used by many laboratories for peptide/protein characterization yet is often considered simple and unglamorous technology. Notably, achieving quantitative accuracy and usable results from high sensitivity amino acid analysis is far from simple. Complications come from a variety of sources including (i) variability in the inherent lability of amino acids to hydrolysis, oxidation and modification; (ii) variability in instrument performance and hydrolysis/derivatization conditions; (iii) background or sample contamination; (iv) data reduction methods; and (v) work habits. Primary emphasis in the tutorial will be placed upon teaching basic techniques to entry level practitioners of amino acid analysis but methodological problems and solutions will be covered in sufficient depth to be informative for the more experienced analyst. Prerequisites, limitations and realistic expectations from current amino acid analysis methods will be emphasized, including strengths and weaknesses of common pre-column and post-column derivatization methods. Special attention will be given to micromethods and improving amino acid analysis background. Sample preparation methods will be discussed including RPHPLC, electrophoresis/electroblotting, precipitation, dialysis, other concentration procedures and useful chemical modifications. Peptide/protein hydrolysis methods and amino acid analysis data reduction methods will be illustrated and instrument maintenance concerns and operating costs highlighted. How to obtain useful amino acid analysis results from limited sample amounts and limited hands-on experience plus when and where to find help will be the reoccurring questions. Tutorial participants will learn through demonstrations and informal discussion. Supported in part by USPHS grants EY06603 and DK38639 and NSF grant DMB 8516111.


Introduction

Methods

Calibration

Hydrolysis

Sample Preparation

Maintenance

How to Reduce Background and Contamination

Performance Overview

Calculations

Identification of Proteins from AAA data

References


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