Toehold mediated strand displacement to measure released product from self-cleaving ribozymes

This paper presents a probe comprising a fluorophore and a quencher, enabling measurement of released product from self-cleaving hammerhead ribozyme, without labeled RNA molecules, regular sampling or use of polyacrylamide gels. The probe is made of two DNA strands; one strand is labelled with a fluorophore at its 5′-end, while the other strand is labelled with a quencher at its 3′-end. These two DNA strands are perfectly complementary, but with a 3′-overhang of the fluorophore strand. These unpaired nucleotides act as a toehold, which is utilized by a detached cleaved fragment (coming from a self-cleaving hammerhead ribozyme) as the starting point for a strand displacement reaction. This reaction causes the separation of the fluorophore strand from the quencher strand, culminating in fluorescence, detectable in a plate reader. Notably, the emitted fluorescence is proportional to the amount of detached cleaved-off RNAs, displacing the DNA quencher strand. This method can replace or complement radio-hazardous unstable 32P as a method of measurement of the product release from ribozyme cleavage reactions; it also eliminates the need for polyacrylamide gels, for the same purpose. Critically, this method allows to distinguish between the total amount of cleaved ribozymes and the amount of detached fragments, resulting from that cleavage reaction.

Variations of Staining Sodium Dodecyl Sulfate–Polyacrylamide Gels with Coomassie Brilliant Blue

Many variations of the original Coomassie Brilliant Blue staining procedure are in use. This protocol describes some selected variations on the standard procedure that give comparable and consistent staining results for proteins in the 20- to 200-kDa range.

Staining Sodium Dodecyl Sulfate–Polyacrylamide Gels with Silver Salts

This protocol describes silver staining procedures to detect low-abundance proteins in sodium dodecyl sulfate-polyacrylamide gels.

Protein Variation Associated With Facial Eczema Resistance or Susceptibility in Romney Sheep

<p>The detection of plasma and liver protein markers for facial eczema resistance or susceptibility in Romney sheep was undertaken. A pooling protocol was used to allow rapid comparison of variation between populations. A 2-D PAGE technigue was developed for protein separation. In general, proteins separated by 2-D PAGE were examined on Coomassie blue or silver stained gels. Greater sensitivity was achieved by labelling proteins with radioactive isotopes. Reductive methylation of the free amino groups of proteins with radioactively labelled formaldehyde and sodium cyanoborohydride was used for isotopic labelling of proteins. A double-labelling technique involving 14C and 3H was used to label plasma or liver proteins from facial eczema resistant and susceptible sheep. The labelled proteins were subsequently separated by 2-D PAGE and detected by autoradiography and fluorography. Any detected variation was further analysed for individuals on one-dimensional polyacrylamide gels which allowed more rapid analysis of multiple samples. No significant difference was detected among the liver proteins of resistant and susceptible sheep. However, among the approximately twenty major plasma protein families visualised on 2-D PAGE gels, significant variation between sheep selected for facial eczema resistance or susceptibility occurred at the transferrin locus. Sheep selected for resistance showed a predominance of acidic transferrins while sheep selected for susceptibility contained a basic transferrin in greater abundance. These results were confirmed and their significance was assessed by transferrin phenotyping on one-dimensional polyacrylamide gels. The transferrin A allele was more abundant in sheep selected for resistance while the transferrin D allele showed a greater association with facial eczema susceptibility. The A allele frequency was 0.57 in resistants and 0.05 in susceptibles while the D allele frequency was 0.18 in resistants and 0.68 in susceptibles. The results suggest some separation of transferrin A and D alleles between the animals selected for resistance and susceptibility. The basis of this variation is unknown. It may reflect either a physiological association of transferrin alleles with a character of importance in facial eczema resistance, or it may be a phenomenon unrelated to facial eczema resistance produced as a result of the way in which the facial eczema resistant and susceptible flocks were generated. It is expected that subsequent genetic studies will show whether transferrin phenotype can be used as a marker to select for facial eczema resistance as a means of controlling the disease.</p>

Protein Variation Associated With Facial Eczema Resistance or Susceptibility in Romney Sheep

<p>The detection of plasma and liver protein markers for facial eczema resistance or susceptibility in Romney sheep was undertaken. A pooling protocol was used to allow rapid comparison of variation between populations. A 2-D PAGE technigue was developed for protein separation. In general, proteins separated by 2-D PAGE were examined on Coomassie blue or silver stained gels. Greater sensitivity was achieved by labelling proteins with radioactive isotopes. Reductive methylation of the free amino groups of proteins with radioactively labelled formaldehyde and sodium cyanoborohydride was used for isotopic labelling of proteins. A double-labelling technique involving 14C and 3H was used to label plasma or liver proteins from facial eczema resistant and susceptible sheep. The labelled proteins were subsequently separated by 2-D PAGE and detected by autoradiography and fluorography. Any detected variation was further analysed for individuals on one-dimensional polyacrylamide gels which allowed more rapid analysis of multiple samples. No significant difference was detected among the liver proteins of resistant and susceptible sheep. However, among the approximately twenty major plasma protein families visualised on 2-D PAGE gels, significant variation between sheep selected for facial eczema resistance or susceptibility occurred at the transferrin locus. Sheep selected for resistance showed a predominance of acidic transferrins while sheep selected for susceptibility contained a basic transferrin in greater abundance. These results were confirmed and their significance was assessed by transferrin phenotyping on one-dimensional polyacrylamide gels. The transferrin A allele was more abundant in sheep selected for resistance while the transferrin D allele showed a greater association with facial eczema susceptibility. The A allele frequency was 0.57 in resistants and 0.05 in susceptibles while the D allele frequency was 0.18 in resistants and 0.68 in susceptibles. The results suggest some separation of transferrin A and D alleles between the animals selected for resistance and susceptibility. The basis of this variation is unknown. It may reflect either a physiological association of transferrin alleles with a character of importance in facial eczema resistance, or it may be a phenomenon unrelated to facial eczema resistance produced as a result of the way in which the facial eczema resistant and susceptible flocks were generated. It is expected that subsequent genetic studies will show whether transferrin phenotype can be used as a marker to select for facial eczema resistance as a means of controlling the disease.</p>

Hydrogels for Three-Dimensional Ionizing-Radiation Dosimetry

Radiation-sensitive gels are among the most recent and promising developments for radiation therapy (RT) dosimetry. RT dosimetry has the twofold goal of ensuring the quality of the treatment and the radiation protection of the patient. Benchmark dosimetry for acceptance testing and commissioning of RT systems is still based on ionization chambers. However, even the smallest chambers cannot resolve the steep dose gradients of up to 30–50% per mm generated with the most advanced techniques. While a multitude of systems based, e.g., on luminescence, silicon diodes and radiochromic materials have been developed, they do not allow the truly continuous 3D dose measurements offered by radiation-sensitive gels. The gels are tissue equivalent, so they also serve as phantoms, and their response is largely independent of radiation quality and dose rate. Some of them are infused with ferrous sulfate and rely on the radiation-induced oxidation of ferrous ions to ferric ions (Fricke-gels). Other formulations consist of monomers dispersed in a gelatinous medium (Polyacrylamide gels) and rely on radiation-induced polymerization, which creates a stable polymer structure. In both gel types, irradiation causes changes in proton relaxation rates that are proportional to locally absorbed dose and can be imaged using magnetic resonance imaging (MRI). Changes in color and/or opacification of the gels also occur upon irradiation, allowing the use of optical tomography techniques. In this work, we review both Fricke and polyacrylamide gels with emphasis on their chemical and physical properties and on their applications for radiation dosimetry.

A rapid method for protein staining in polyacrylamide gels using water saturated with chloroform

Polyacrylamide gel electrophoresis, followed by an appropriate staining, is a popular and useful analytical procedure for protein identification and characterization. The aim of this study was to develop a method for protein visualization in polyacrylamide gels that would be alternative to Coomassie blue or silver staining. The proposed method is simple, fast and inexpensive. The optimized protocol for protein staining and visualization takes as little as 6 minutes and utilizes deionized water and chloroform. Fluorescence of proteins is induced by UV light and can be detected with a standard transilluminator.

External morphological stages and protein variations along with the embryonic development of the longarm river prawn Macrobrachium tenellum (Smith, 1871)

A good understanding of a given species' embryology is important to settle the larval rearing bases when juveniles are required for culture purposes or conservation programs. Changes in embryonic morphology, protein concentration, and protein type occurring in prawn eggs were analyzed in the present work. Berried females of Macrobrachium tenellum were collected in the Colotepec River, Oaxaca, Mexico. The eggs were taken from the ovigerous mass and embryonic stages classified by their color. Morphological changes in the embryos allowed identifying six embryonic stages based on color, egg size, and morphological features. Determinations of the protein extract were executed in SDS-PAGE (electrophoresis in polyacrylamide gels) and, subsequently, proteomic analyses were also performed. Protein bands along embryonic development and their molecular weights are presented and commented.