Mikroelektrophorese in kontinuierlichen Polyacrylamid- Gradientengelen, III^[1,2]. Extraktion und Fraktionierung von Ribonucleinsäuren im Mikromaßstab

Abstract

Zusammenfassung: Kontinuierliche PolyacrylamidGradientengele von l -50% in l-, 2-, 5-und 10--Kapillaren sind zur Fraktionierung von 4S-28S-RNA geeignet. Es werden verschiedene Gel-und Puffersysteme für spezielle Trennprobleme, verschiedene Färbemethoden und Mikroextraktionsverfahren zur Präparation der RNA aus l -5 mg Gewebe angegeben. Für eine Elektrophorese genügen ca. 0.1 g einer RNA-Mischung. Kontinuierliche Puffersysteme sind besonders zur Fraktionierung hochmolekularer und diskontinuierliche zur Trennung niedermolekularer RNA geeignet. Im steilen Gelgradienten können auch kleine RNAFragmente bis zum Molekulargewicht 3000 fraktioniert werden. Der Nachweis von Konformationsänderungen ist ebenfalls möglich. Mikrogradienten mit flacher Steigung können zur Darstellung von Ribosomen und ihren Untereinheiten verwandt werden. Micro-electrophoresis in continuous polyacrylamide gradient gels, HI. Extraction and fractionation of ribonucleic acids on micro scaleSummary: Polyacrylamide gels with continuous gradients ranging from 1 -50 % were prepared in 1, 2, 5 and 10-capillaries and were found suitable for fractioning 4S-28S RNA. Different gel and buffer systems for special fractionation problems and several staining procedures are described as well as methods to prepare RNA from 1-5 mg tissue samples. For the electrophoresis of a mixture of RNA approximately 0.1 g is sufficient. Continuous electrophoretic conditions are preferentially used for high molecular weight RNA, whereas discontinuous electrophoresis is suitable for RNA species of low molecular weight. Applying gels with steep gradients (1-50%), fragments of RNA with molecular weights as low as 3000 can be separated. Moreover, conformational changes can be shown. Ribosomes and their subunits can be fractionated on micro gradient gels of acrylamide concentrations ranging from 1 -15 %.

Separation of aminoacyl‐tRNAPhe and tRNAsPhe with partially hydrolysed 3′‐end by polyacrylamide gradient micro‐electrophoresis

“…It was shown that the E. coli ribosomal 5s RNA is able to undergo conformational changes, which can be detected by gel electrophoresis [9,14]. It is therefore possible that the observed unusual electrophoretical behavior of the tRNA molecules missing their 3'-end is also due to a change in the overall shape of the molecule.…”

“…Concentration of gradients was l-40% monomer at 2% crosslinking. As gel buffer 350 mM Tris-HzS04, pH 8.4 and as electrophoresis buffer 50 mM Tris-glycin, pH 8.4 was used [9] . Samples were applied on to the gel in quantities of 0.2-0.7 pmoles tRNA.…”

“…were obtained using the Joyce-Loeble doublebeam microdensitometer with 100: 1 ratio of record to sample travel using a gray wedge of 0.79 d. Further details about the micro-electrophoresis on polyacrylamide gradient gels are presented elsewhere [7,8,9,13].…”

Tyrosyl-tRNA Synthetase from Baker's Yeast. Rapid Isolation by Affinity Elution, Molecular Weight of the Enzyme, and Determination of Essential Sulfhydryl Groups

“…An approximate value of 1 A260 unit corresponding to 1.5 nmol of tRNA was used for calculations. The purity of tRNATy' was examined by polyacrylamide gradient gel electrophoresis according to Neuhoff [19] and on the basis of specific aminoacylation. The purification of tRNATy' in these three chromatographic steps is summarized in Table 1.…”

Differenzierung der Proteinurie durch die Mikroelektrophorese in kontinuierlichen Polyacrylamid-Gradientengelen

“…Hier soll die Anwendung einer Methode (Rfichel et al [13,14], Wolfrum et al [17], Neuhoff [11]) dargestellt werden, die als Mikromethode einige Vorteile hinsichtlich des methodischen, zeitlichen und finanziellen Aufwandes besitzt und die in ihren Ergebnissen vergleichbaren Verfahren ebenbih'tig erscheint.…”