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Sequential purification of human apolipoprotein B-100, albumin, and fibrinogen by immunoaffinity chromatography for measurement of protein synthesis.
Anal Biochem. 1997 May 01; 247(2):228-36.AB

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 samples collected during a 10-h primed continuous intravenous infusion of L-[1-13C]leucine confirmed that this technique provides an efficient way to purify plasma VLDL ApoB-100, albumin, and fibrinogen for measuring their synthetic rates in human metabolism studies.

Authors+Show Affiliations

Mayo Clinic, Rochester, Minnesota 55905, USA.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

9177682

Citation

Fu, A Z., et al. "Sequential Purification of Human Apolipoprotein B-100, Albumin, and Fibrinogen By Immunoaffinity Chromatography for Measurement of Protein Synthesis." Analytical Biochemistry, vol. 247, no. 2, 1997, pp. 228-36.
Fu AZ, Morris JC, Ford GC, et al. Sequential purification of human apolipoprotein B-100, albumin, and fibrinogen by immunoaffinity chromatography for measurement of protein synthesis. Anal Biochem. 1997;247(2):228-36.
Fu, A. Z., Morris, J. C., Ford, G. C., & Nair, K. S. (1997). Sequential purification of human apolipoprotein B-100, albumin, and fibrinogen by immunoaffinity chromatography for measurement of protein synthesis. Analytical Biochemistry, 247(2), 228-36.
Fu AZ, et al. Sequential Purification of Human Apolipoprotein B-100, Albumin, and Fibrinogen By Immunoaffinity Chromatography for Measurement of Protein Synthesis. Anal Biochem. 1997 May 1;247(2):228-36. PubMed PMID: 9177682.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Sequential purification of human apolipoprotein B-100, albumin, and fibrinogen by immunoaffinity chromatography for measurement of protein synthesis. AU - Fu,A Z, AU - Morris,J C, AU - Ford,G C, AU - Nair,K S, PY - 1997/5/1/pubmed PY - 1997/5/1/medline PY - 1997/5/1/entrez SP - 228 EP - 36 JF - Analytical biochemistry JO - Anal. Biochem. VL - 247 IS - 2 N2 - 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 samples collected during a 10-h primed continuous intravenous infusion of L-[1-13C]leucine confirmed that this technique provides an efficient way to purify plasma VLDL ApoB-100, albumin, and fibrinogen for measuring their synthetic rates in human metabolism studies. SN - 0003-2697 UR - https://www.unboundmedicine.com/medline/citation/9177682/Sequential_purification_of_human_apolipoprotein_B_100_albumin_and_fibrinogen_by_immunoaffinity_chromatography_for_measurement_of_protein_synthesis_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0003-2697(97)92086-8 DB - PRIME DP - Unbound Medicine ER -