Separation of ribonucleic acids by polyacrylamide gel electrophoresis

doi:10.1016/0003-2697(68)90321-7

Analytical Biochemistry

Volume 22, Issue 2, February 1968, Pages 311-320

https://doi.org/10.1016/0003-2697(68)90321-7 Get rights and content

Abstract

1.
1. A method is described for the fractionation of RNA from whole tissue by acrylamide gel electrophoresis in a discontinuous buffer system of pH 5.5–7.5. The highest resolution was achieved when the sample migrated through layers of gels of increasing acrylamide concentration and correspondingly decreasing pore size.
2.
2. RNA fractions in acrylamide gel were stained with gallocyaninchromalum. Relative amounts were determined by densitometry. With E. coli sRNA or yeast RNA, the RNA-dye complex in acrylamide gel was shown to follow Beer's law in the range between 0.4 and 1.7 mg RNA/ml.
3.
3. In addition to the two ribosomal and the soluble RNA fractions, several minor components were regularly found in RNA extracted from Drosophila and Chironomus tentans by the phenol-SDS method. These could not be detected in the UV absorbancy pattern of sucrose gradients of the same preparations.

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The culture filtrate of Mycobacterium tuberculosis H37Ra was fractionated by ammonium sulphate precipitation, diethyl aminoethyl (DEAE) cellulose-Sephadex A-50 column chromatography, preparative isoelectic focusing and polyacrylamide gal electrophoresis to isolate the most antigenic tuberculoprotein. One protein, designated P2, was isolated and determined by immunodiffusion to be the most immunologically reactive antigen present in H37Ra culture filtrate. This protein had a molecular weight of about 22 000, a sedimentation constant of 2.2S and was present in Seibert's A, B, and C proteins.
Le filtrat de culture de M, tuberculosis H37Ra a été fractionné par précipitation par le sulfate d'ammonium, par Chromatographie sur colonne Sephadex A-50 de diéthyl aminoéthyl de cellulose (DEAE) ‘préparative’ iso-electro-focusing et électrophorèse sur gel de polyacrylamide afin d'isoler les tuberculoprotéines les plus antigéniques Une des protéines, appelée P2, a été isolée et définie, par immunodiffusion, comme étant l'antigène le plus immunologiquement réagissant présent dans le filtrat de culture de H37Ra. Le poids moléculaire de cette protéine était de 22 000, sa constante de sédimentation était de 2,2 S et elle était présente dans les protéines A, B et C de Seibert.
El filtrado de un cultivo de Mycobacterium tuberculosis H 37 Ra fue fraccionado mediante precipitación por sulfato de amonio, cromatografia en columna de Sephadex A-50-dietil aminoetil (DEAE) celulosa, isoelectrofocalización preparativa y electro- foresis preparativa en gel de poliacrilamida, con el objeto de aislar la proteina tuberculosa más antigénica. Se aisló una proteina llamada P2 y medinate inmuno- difusión se demostró que P2 era el antigeno inmunológicamente más reactivo presente en el filtrado del cultivo H 37 Ra. Esta proteina tiene un peso molecular de approximadamente 22 000, una constante de sedimentación de 2,2S y está presente en las proteinas A, B y C de Seibert.
Chapter 3 Staining of RNA after Polyacrylamide Gel Electrophoresis
1975, Methods in Cell Biology
This chapter highlights the staining techniques for RNA after polyacrylamide gel electrophoresis with emphasis on fluorescence detection method. One of the critical steps in polyacrylamide gel electrophoresis is detection of the substance analyzed—electrophoresed RNA. Staining appears to be the simplest and a very sensitive method of RNA detection. The requirements for RNA staining techniques include ability to make possible the visualization of very small amounts of RNA (0.01 μg and less) and minimal binding of the stain to the gel (minimal background). Fixatives of any type induce changes in the physical state of the RNA molecule. This applies to both fixatives in which the metal component is linked to the RNA molecule and which form stronger bonds, the changes induced thus being more persistent, and fixatives that are active without being bound to RNA in which the changes induced persist only as long as the RNA remains in such fixatives. The purpose of all these changes is to induce stronger binding of the dye to the RNA. Fixative stain solutions are used exclusively for RNA staining—these contain, in addition to the dye, one or a combination of several fixatives. Loening's method is most suitable for fractionation of all RNA species, with perspective of both the purity of the gel and the possibility of densitometric evaluation of RNA fractions—either directly or after staining.
Electrophoretic separation of enzymes of individual flour beetles, Tribolium castaneum, on polyacrylamide gel
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The use of a new buffer solution for electrophoresis of the dsRNA of wound tumor virus permitted the electrophoretic separation and identification of all 12 ds RNA components of WTV RNA. The molecular weight of the genome was estimated to be 15.06 × 10⁶ daltons. All 10 components of reovirus type 2 could be separated by use of the same solution. The electrophoretic patterns for the first 7 components of wound tumor virus and rice dwarf virus are very similar.
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Eighteen human fibroblast strains were tested for mycoplasma contamination by polyacrylamide gel electrophoresis of ³H-uridine labeled RNA and by standard microbiological culture techniques. Despite negative culture results, prominent 23S and 16S RNA peaks were detected in 11 of these cell strains. The mycoplasma origin of this RNA was indicated by electron microscopic demonstration of these organisms. Another indication of contamination was the decreased specific radioactivity of whole cell RNA extracted from cell strains infected with mycoplasma.
Conformational effects of mercurials on rat liver ribosomes: A comparison between the unmasking of a shielded protein in the 60S subunit by phenyl mercurials and edta
1973, Chemico-Biological Interactions
- 1.
  (1) Treatment of rat liver ribosomes with the mercurials p-hydroxymercuribenzoate (PHMB) or p-chloromercuriphenyl sulfonate (PCMPS), gives rise to characteristic alterations in the electrophoretic pattern of the proteins, indicating a binding of the reagents in situ to certain protein components. The reaction is reversed by mercaptoethanol or dithiothreitol (DTT).
- 2.
  (2) In the presence of 5–15 mM MgCl₂ (or lower concentrations of Ca²⁺ or Mn²⁺) the reaction with PHMB and PCMPS gives rise to a conformational alteration, by which a normally shielded protein in the large subunit (‘protein 5’) is made susceptible to chymotrypsin. The unmasking is reversed by mercaptoethanol or DTT, but cannot be effectively prevented by pretreatment of the ribosomes with SH-blocking reagents such as iodoacetate or N-ethylmaleimide (NEM).
- 3.
  (3) The activity of the ribosomes with respect to endogenous or poly U-directed phenylalanine incorporation is strongly inhibited by treatment with PHMB or PCMPS under the conditions of unmasking, but can be restored to a great extent with mercaptoethanol or DTT. During the period of mercurial-dependent unmasking the ribosomes are abnormally sensitive to inactivation by chymotrypsin, suggesting a functional importance of the unmasked chain(s).
- 4.
  (4) Reversible dissociation by current methods does not lead to the unmasking of protein 5. (Reversible dissociation by increased ionic strength leads to the unmasking of another protein in the 60S subunit, ‘protein 10’.) In contrast to the Mg²⁺-stimulated unmasking by PHMB or PCMPS, the unmasking by Mg²⁺ deprivation (ethylenediaminetetraacetate, EDTA) is irreversible. Although treatment of the ribosomes with PHMB or PCMPS under the conditions of unmasking of protein 5 may involve some dissociation of ribosomes to subunits, the unmasking is not dependent on this reaction.
- 5.
  (5) Neither the Mg²⁺ stimulated unmasking of proteins by PHMB or PCMPS, nor the unmasking by Mg²⁺-deprivation (EDTA) depends on RNA degradation or detachment of 5S RNA. The striking (although not complete) similarity between the two patterns of unmasking lends further support to the notion that the large subunit in mammalian ribosomes is predisposed to certain types of basic conformational reactions, which can be initiated by alternative and seemingly unrelated mechanisms.

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¹: Career Scientist of the Health Research Council of the City of New York (I-190).

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Separation of ribonucleic acids by polyacrylamide gel electrophoresis

Abstract

Anal Biochem

Biochim. Biophys. Acta

J. Mol. Biol

Ann. N. Y. Acad. Sci

Anal. Biochem

Biochim. Biophys. Acta

Ann. N. Y. Acad. Sci

Ann. N. Y. Acad. Sci