Sequential purification of human apolipoprotein B-100, albumin, and fibrinogen by immunoaffinity chromatography for measurement of protein synthesis

Analytical Biochemistry
A Z FuK S Nair

Abstract

A determinant of the accuracy of protein synthesis measurement using stable isotope is the purity of the protein under study. An Immunoaffinity chromatographic technique to sequentially purify human plasma albumin, fibrinogen, and apolipoprotein B-100 (ApoB-100) was developed to measure isotopic enrichment in these proteins. The technique, utilizing immobilized mouse monoclonal antibodies specific to human plasma ApoB-100, albumin, and fibrinogen onto an affinity matrix, allowed purification of very low density lipoprotein (VLDL) ApoB-100, albumin, and fibrinogen from 1- to 2-ml plasma samples. Analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining demonstrated consistent purity of the three purified proteins. The identity and the purity of the proteins separated by this technique were also confirmed by amino acid sequence analysis. This technique was applied to sequentially purify and measure the isotopic enrichment in those proteins by mass spectrometry from human plasma samples collected after orally ingesting L[1-13C]-leucine. Reproducibility of the enrichment measurements is within 5% of the coefficient of variation. Measurements [13C]leucine in these proteins purified from plasma ...Continue Reading

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Citations

Jul 28, 2010·Experimental Animals·Makoto KinoshitaTamio Teramoto
Dec 25, 2003·American Journal of Physiology. Endocrinology and Metabolism·Stephen F PrevisBernard R Landau
May 14, 2004·American Journal of Physiology. Endocrinology and Metabolism·K Sreekumaran NairSreekumar Raghavakaimal
Mar 27, 2002·Hepatology : Official Journal of the American Association for the Study of Liver Diseases·Michael CharltonK Sreekumaran Nair
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Feb 2, 2006·American Journal of Physiology. Endocrinology and Metabolism·Abdul JaleelK Sreekumaran Nair
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Dec 9, 1998·The American Journal of Physiology·A Fu, K S Nair

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