Evaluation of SYPRO Ruby total protein stain for the normalization of two-dimensional Western blots

Due to post-translational modifications such as phosphorylation, proteins exist as distinct charge variants. Two-dimensional (2D) gel electrophoresis followed by immunoblotting enables the detection of these isoforms. For their accurate relative quantitation in different samples, a loading control is necessary to compensate for technical errors such as imprecise sample loading or transfer. The study reveals that the combinatory approach of SYPRO Ruby and chemiluminescence-based 2D Western blot analysis exhibits high linearity and excellent reproducibility and is applicable for limited sample amounts.     

Cloning,characterization and localization of <Emphasis Type="Italic">CHS</Emphasis> gene from blood orange, <Emphasis Type="Italic">Citrus sinensis</Emphasis> (L.) Osbeck cv. Ruby

Chalcone synthase (CHS) is involved in the biosynthesis of anthocyanin. In this study, a full-length DNA of CHS gene (named as CsCHS-bo) was cloned from the blood orange, Citrus sinensis (L.) Osbeck cv. Ruby. The gene was 1,512 bp in size containing an open reading frame (1,176 bp) encoding 391 amino acids. Comparative and bioinformatic analyses revealed that the deduced protein of CsCHS-bo was highly homologous to CHS from other plant species. The protein of CsCHS-bo had four CHS-specific conserved motifs and a CHS-family signature sequence GFGPG. Phylogenetic analysis indicated that the protein of CsCHS-bo was in a subgroup with CHS of Ruta Palmatum. The CsCHS-bo was localized to the chromosomes 2p, 4p and 6p by an improved fluorescence in situ hybridization technique, indicating that at least three copies of CsCHS-bo were present in the genome. The novel nucleotide sequence data published here have been deposited in the EMBL/DDBJ/GenBank databases under accession number EU410483.     

Identification of Endothelial Proteins by MALDI-MS Using a Compact Disc Microfluidic System

Vascular endothelial proteins have been analyzed using two-dimensional (2D) gel electrophoresis and subsequent mass spectrometry, with separate methods for the intervening sample preparations. Compact disc (CD) technology was found to be rapid, giving high overall yield both with ordinary Coomassie staining and with Sypro Ruby staining. Combined with automatic in-gel digestion, the CD technology has great capacity for large numbers of protein analysis, although for limited sample numbers, manual methods can give similar sequence coverage. In a test set of 48 samples, 45 proteins were identified using the CD preparation technique, 32 identified with higher sequence coverage using the CD technique, 7 with higher using ZipTips in a robotic workstation, and 5 with higher coverage using dried droplets of unpurified samples. In the process of these methodological comparisons, basic patterns for 116 endothelial proteins were defined, representing 297 separate protein spots on the 2D gels.     

Comparison of DIGE and post-stained gel electrophoresis with both traditional and SameSpots analysis for quantitative proteomics

2-DE is an important tool in quantitative proteomics. Here, we compare the deep purple (DP) system with DIGE using both a traditional and the SameSpots approach to gel analysis. Missing values in the traditional approach were found to be a significant issue for both systems. SameSpots attempts to address the missing value problem. SameSpots was found to increase the proportion of low volume data for DP but not for DIGE. For all the analysis methods applied in this study, the assumptions of parametric tests were met. Analysis of the same images gave significantly lower noise with SameSpots (over traditional) for DP, but no difference for DIGE. We propose that SameSpots gave lower noise with DP due to the stabilisation of the spot area by the common spot outline, but this was not seen with DIGE due to the co-detection process which stabilises the area selected. For studies where measurement of small abundance changes is required, a cost-benefit analysis highlights that DIGE was significantly cheaper regardless of the analysis methods. For studies analysing large changes, DP with SameSpots could be an effective alternative to DIGE but this will be dependent on the biological noise of the system under investigation.     

锦带花属2个新品种

描述了2个锦带花属新品种,即贵妃锦带（Weigela florida ‘Royal’）和宝石锦带(Weigela florida‘Ruby’)。二者相近似,区别在于贵妃锦带植株稍高,株高1.5～2.0 m,花为尖晶玉红色,叶色暗绿;宝石锦带植株较矮,株高1.0～1.6 m,花为丹紫红色,叶片金黄色。这2个新品种均具有观赏特异性,抗逆性强,花期早且长等特性。     

Range dynamics of small mammals along an elevational gradient over an 80‐year interval

One expected response to observed global warming is an upslope shift of species elevational ranges. Here, we document changes in the elevational distributions of the small mammals within the Ruby Mountains in northeastern Nevada over an 80‐year interval. We quantified range shifts by comparing distributional records from recent comprehensive field surveys (2006–2008) to earlier surveys (1927–1929) conducted at identical and nearby locations. Collector field notes from the historical surveys provided detailed trapping records and locality information, and museum specimens enabled confirmation of species' identifications. To ensure that observed shifts in range did not result from sampling bias, we employed a binomial likelihood model (introduced here) using likelihood ratios to calculate confidence intervals around observed range limits. Climate data indicate increases in both precipitation and summer maximum temperature between sampling periods. Increases in winter minimum temperatures were only evident at mid to high elevations. Consistent with predictions of change associated with climate warming, we document upslope range shifts for only two mesic‐adapted species. In contrast, no xeric‐adapted species expanded their ranges upslope. Rather, they showed either static distributions over time or downslope contraction or expansion. We attribute these unexpected findings to widespread land‐use driven habitat change at lower elevations. Failure to account for land‐use induced changes in both baseline assessments and in predicting shifts in species distributions may provide misleading objectives for conservation policies and management practices.     

Quantitative proteomics: assessing the spectrum of in-gel protein detection methods

Proteomics research relies heavily on visualization methods for detection of proteins separated by polyacrylamide gel electrophoresis. Commonly used staining approaches involve colorimetric dyes such as Coomassie Brilliant Blue, fluorescent dyes including Sypro Ruby, newly developed reactive fluorophores, as well as a plethora of others. The most desired characteristic in selecting one stain over another is sensitivity, but this is far from the only important parameter. This review evaluates protein detection methods in terms of their quantitative attributes, including limit of detection (i.e., sensitivity), linear dynamic range, inter-protein variability, capacity for spot detection after 2D gel electrophoresis, and compatibility with subsequent mass spectrometric analyses. Unfortunately, many of these quantitative criteria are not routinely or consistently addressed by most of the studies published to date. We would urge more rigorous routine characterization of stains and detection methodologies as a critical approach to systematically improving these critically important tools for quantitative proteomics. In addition, substantial improvements in detection technology, particularly over the last decade or so, emphasize the need to consider renewed characterization of existing stains; the quantitative stains we need, or at least the chemistries required for their future development, may well already exist.     

Comparison of SYPRO Ruby and Flamingo fluorescent stains for application in proteomic research

Fluorescent dyes are widely used for the detection and quantitation of proteins separated by polyacrylamide gel electrophoresis. SYPRO Ruby is one such fluorescent dye widely used for this purpose. More recently, another fluorescent dye, Flamingo, is available for expression proteomic research. Using a standard ultraviolet (UV) transilluminator and a charge-coupled device (CCD)-based imaging system, the relative sensitivity of these two different fluorescent stains with regard to detection of protein spots separated by two-dimensional gel electrophoresis (2D-GE) and identification by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) were compared. Using mouse kidney and liver homogenates as well as Escherichia coli extract, we detected a greater number of protein spots using Flamingo compared with SYPRO Ruby. In addition, when we compared the number of matched peptides and the percentage of amino acid residues identified for 22 different protein spots of mouse kidney proteome, we observed a higher number of matched peptides and a higher percentage of amino acid residues for the majority of the proteins using Flamingo compared with SYPRO Ruby. Also, we were able to characterize a protein spot that can be detected by Flamingo only. Therefore, we recommend Flamingo over SYPRO Ruby to be used for studies on expression proteomics.