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Titolo:
Comparison of yeast cell protein solubilization procedures for two-dimensional electrophoresis
Autore:
Harder, A; Wildgruber, R; Nawrocki, A; Fey, SJ; Larsen, PM; Gorg, A;
Indirizzi:
Techol,iv Munich, Lehrstuhl Allgemeine Lebensmitteltechnol, Dept Food Techn Tech Univ Munich Freising Germany D-85350 mitteltechnol, Dept Food Techn Ctr Proteome Anal Life Sci, Odense, Denmark Ctr Proteome Anal Life Sci Odense Denmark nal Life Sci, Odense, Denmark
Titolo Testata:
ELECTROPHORESIS
fascicolo: 4-5, volume: 20, anno: 1999,
pagine: 826 - 829
SICI:
0173-0835(199904/05)20:4-5<826:COYCPS>2.0.ZU;2-9
Fonte:
ISI
Lingua:
ENG
Soggetto:
IMMOBILIZED PH GRADIENTS; 2-DIMENSIONAL ELECTROPHORESIS; CURRENT STATE;
Keywords:
immobilized pH gradient; Saccharomyces cerevisiae; two-dimensional polyacrylamide gel electrophoresis; yeast protein solubilization;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
12
Recensione:
Indirizzi per estratti:
Indirizzo: Gorg, A Techol,iv Munich, Lehrstuhl Allgemeine Lebensmitteltechnol, Dept Food Techn Tech Univ Munich Freising Germany D-85350 chnol, Dept Food Techn
Citazione:
A. Harder et al., "Comparison of yeast cell protein solubilization procedures for two-dimensional electrophoresis", ELECTROPHOR, 20(4-5), 1999, pp. 826-829

Abstract

Three different procedures for the solubilization of yeast (S. cerevisiae)cell proteins were compared on the basis of the obtained two-dimensional (2-D) polypeptide patterns. Major emphasis was laid on minimizing handling steps, protein modification or degradation, and quantitative loss of high molecular mass proteins. The procedures employed were sonication, followed by(i) protein solubilization with "standard" lysis buffer (9 M urea, 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) (CHAPS), 1% dithiothreitol (DTT), 2% v/v carrier ampholytes, (ii) presolubilization of proteinswith sodium dodecyl sulfate (SDS) buffer, consisting of 1% SDS and 100 mM tris(hydroxymethyl)aminomethane (Tris)-HCl, pH 7.0, followed by dilution with "standard" lysis buffer, and (iii) boiling the sample with SDS during cell lysis, followed by dilution with thiourea/urea lysis buffer (2 bn thiourea/7 M urea, 4% w/v CHAPS, 1% w/v DTT, 2% v/v carrier ampholytes). All procedures tested were rapid and simple. However, with the first procedure (i),considerable degradation of high M-r proteins occurred. In contrast, protein degradation was minimized by boiling the sample in SDS buffer immediately after sonication (method ii). Protein disaggregation and solubilization of high M-r proteins were further improved by pre-boiling with SDS and usingthiourea/urea lysis buffer instead of "standard" lysis buffer (procedure iii).

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Documento generato il 29/03/20 alle ore 15:39:10