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Current Topics in Analytical Chemistry   Volumes    Volume 2 
Abstract
Consecutive sodium dodecyl sulphate polyacrylamide gel electrophoresis and reversed phase high performance liquid chromatography. Applications to the micro-purification and analysis of proteins, peptides and related compounds
Batia Kaplan
Pages: 159 - 169
Number of pages: 11
Current Topics in Analytical Chemistry
Volume 2 

Copyright © 2001 Research Trends. All rights reserved

ABSTRACT

A procedure combining the electrophoretic and chromatographic techniques was developed by us for small-scale purification of proteins and peptides. In contrast to other commonly used combined methods, this developed technique includes analytical-scale slab gel SDS-electrophoresis, as a first micro-preparative separation step. Depending on the nature, molecular mass and quantity of the electrophoretically separated macromolecules, they are either eluted from the gel and then purified from SDS, or transferred to PVDF membranes and then eluted. These procedures were optimized and adapted for the subsequent reversed phase HPLC. The developed purification technique was applied to different fields of biomedical research and was fruitful in obtaining a high degree of purity of the isolated material needed for its chemical characterization. This included the studies on different amyloid proteins (4 - 17kDa) deposited in body tissues of patients with systemic amyloidosis and Alzheimer`s disease; analysis of the isomeric forms of acute phase reactants - serum amyloid A proteins (12kDa) in the inflamed plasma of mice; search for middle sized molecules (<2kDa) accumulating in the uremic sera of hemodialysis patients Our micro-technique combines the resolution power of analytical gels with high separation speed of HPLC, and appears to represent a useful tool in biochemical studies of small biological samples.

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