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Titolo:
Proteomics of rat liver Golgi complex: Minor proteins are identified through sequential fractionation
Autore:
Taylor, RS; Wu, CC; Hays, LG; Eng, JK; Yates, JR; Howell, KE;
Indirizzi:
Univ Colorado, Sch Med, Dept Cellular & Struct Biol, Denver, CO 80262 USA Univ Colorado Denver CO USA 80262 lar & Struct Biol, Denver, CO 80262 USA Univ Washington, Hlth Sci Ctr, Dept Mol Biotechnol, Seattle, WA 98195 USA Univ Washington Seattle WA USA 98195 ol Biotechnol, Seattle, WA 98195 USA
Titolo Testata:
ELECTROPHORESIS
fascicolo: 16, volume: 21, anno: 2000,
pagine: 3441 - 3459
SICI:
0173-0835(200010)21:16<3441:PORLGC>2.0.ZU;2-O
Fonte:
ISI
Lingua:
ENG
Soggetto:
GTP-BINDING PROTEIN; TRANSITIONAL ENDOPLASMIC-RETICULUM; 2-DIMENSIONAL GEL-ELECTROPHORESIS; INTEGRAL MEMBRANE-PROTEIN; CELL-FREE SYSTEM; TRANSPORT VESICLES; SECRETORY PATHWAY; CIS-GOLGI; POLYACRYLAMIDE GELS; EXOCYTIC TRANSPORT;
Keywords:
cycloheximide; Golgi complex; proteomics; Triton X-114; two-dimensional gel electrophoresis;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
66
Recensione:
Indirizzi per estratti:
Indirizzo: Howell, KE Univ Colorado, Sch Med, Dept Cellular & Struct Biol, Box B-111,4200 E 9th Ave, Denver, CO 80262 USA Univ Colorado Box B-111,4200 E 9th AveDenver CO USA 80262 USA
Citazione:
R.S. Taylor et al., "Proteomics of rat liver Golgi complex: Minor proteins are identified through sequential fractionation", ELECTROPHOR, 21(16), 2000, pp. 3441-3459

Abstract

The discovery of novel proteins resident to the Golgi complex will fuel our future studies of Golgi structure/function and provide justification for proteomic analysis of this organelle. Our approach to Golgi proteomics was to first isolate and characterize the University of Colorado, intact organelle free of proteins in transit by use of tissue pretreated with cycloheximide. Then the stacked Golgi fraction was fractionated into biochemically defined subfractions: Triton X-114 insoluble, aqueous, and detergent phases. The aqueous and Department of Molecular detergent phases were further fractionated by anion-exchange column chromatography. In addition, radiolabeled cytosol was incubated with stacked Golgi fractions containing proteins in transit, and the proteins bound to the Golgi stacks in an energy-dependent manner were characterized. All fractions were analyzed by two-dimensional (2-D) gel electrophoresis and identification numbers were given to 588 unique2-D spots. Tandem mass spectrometry was used to analyze 93 of the most abundant 2-D spots taken from preparative Triton X-114 insoluble, aqueous and detergent phase 2-D gels. Fifty-one known and 22 unknown proteins were identified. This study represents the first installment in the mammalian Golgi proteome database. Our data suggest that cell fractionation followed by biochemical dissection of specific classes of molecules provides a significantadvantage for the identification of low abundance proteins in organelles.

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Documento generato il 20/09/20 alle ore 06:39:37