Postelectrophoretic staining of proteins separated by two-dimensional gel electrophoresis using SYPRO dyes

While the classical silver stain has been the method of choice for high sensitivity protein visualization on two-dimensional gel electrophoresis (2-D PAGE), post-electrophoretic fluorescent staining with the SYPRO group of dyes has emerged to challenge silver staining for proteome analysis. The latter offers improved sensitivity, higher dynamic range and easy handling. However, most of the published data were derived from analysis of 1-D gel separations. In this work, we have focused on three commercially available fluorescent dyes, SYPRO Ruby, SYPRO Orange and SYPRO Red (Molecular Probes, Eugene, OR, USA) and studied their sensitivity and dynamic range on 2-D PAGE. The use of a multiwavelength fluorescent scanner to image 2-D protein profiles visualized with fluorescent staining is discussed, and a detailed comparison with analysis by silver staining is also provided. These results demonstrate the advantages of using SYPRO dyes, which are in agreement with the literature based on 1-D gel electrophoresis, and give a more realistic understanding of the performance of these fluorescent dyes with 2-D PAGE.     

循环伏安法研究光谱增感染料在溴化银上的吸附

用循环伏安法和电子显微镜研究了不同类型的染料在溴化银上的吸附,使用了电沉积在铂上的溴化银电极作为照相乳剂的模型,证明了由于染料电荷的不同,其在AgBr/Br^-表面上的吸附也不同,正性染料被吸附时,形成难溶的表面络合物,和负性染料相作用时,形成易溶的表面络合物,而当AgBr与非菁负性染料作用时,则由于负电排斥而不形成络合物。因此,利用循环伏安法可以判断不同染料的吸附情况。本文还研完了超增感及Riester提出的‘强色增感’中的吸附情况。实验结果表明在AgBr上染料的排列次序取决于其吸附强度。本工作还证明了Reister所提出的‘强色增感剂’的作用机理是可信的。     

Chemical and biological treatment of waste water with a novel silver/ordered mesoporous alumina nanocomposite

One of the serious problems in the present century is chemical and biological pollution of the environment. Nanocomposites are multiphase solid materials that have been used as adsorbent of pollutants such as dyes, pesticides, anions and etc. in the last decades. In this study, a novel nanocomposite including silver nanoparticles and ordered mesoporous alumina (OMA) has been synthesized and used for the removal of dyes pollutants (methyl orange, bromothymol blue and reactive yellow) from aqueous solution. The characterization of synthesized nanocomposite has been performed by TEM, N₂ adsorption/desorption, XRD and energy dispersive X-ray spectroscopy analysis. The adsorption kinetic and equilibrium data have been obtained by UV–vis spectroscopy. The results show that the silver/OMA nanocomposite (Ag/OMA nanocomposite) is good adsorbent for the removal of anionic dyes from aqueous solution, and also this nanocomposite has a biocidal action against both Gram-negative and Gram-positive bacteria.     

Highly sensitive and simple fluorescence staining of proteins in sodium dodecyl sulfate-polyacrylamide-based gels by using hydrophobic tail-mediated enhancement of fluorescein luminescence

Fluorescein has an extremely low luminescence intensity in acidic aqueous media. However, when it was bound to proteins, subsequent increase of luminescence intensity took place. Furthermore, when a hydrophobic tail, such as aliphatic hydrocarbons, was introduced to fluorescein, more dramatic increase of luminescence intensity was observed upon binding to proteins. In the present study, by utilizing this luminescence enhancement, three hydrophobic fluorescein dyes (5-dodecanoyl amino fluorescein, 5-hexadecanoyl amino fluorescein, and 5-octadecanoyl amino fluorescein) were examined as noncovalent fluorescent stains of protein bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Effective incorporation of the dyes to proteins in gels was accomplished either simply by adding dyes at the protein fixation step, or by treating gels with a staining solution after the fixation. The sensitivity of this staining method using the fluorescein derivatives was approximately 1 ng/band for most proteins. For some cases, protein bands containing as low as 0.1 ng were successfully visualized. In addition, the detection sensitivity showed much less protein-to-protein variation than silver staining. This new staining method was also successfully applied to two-dimensional electrophoresis of rat brain proteins. Its overall sensitivity was comparable to that of silver staining.     

Stain efficiency and MALDI-TOF MS compatibility of seven visible staining procedures

Visible stain is still the most popular protein staining method used in proteomic approaches. However, most published data have been derived from comparisons between visible dyes and fluorescent dyes. In this work, we have focused on seven widely used visible staining procedures—Neuhoff CCB, blue silver, and five silver stains (LKB SN, He SN, Yan SN, Vorum SN, and Blum SN)—and studied their stain efficiencies and MALDI-TOF MS compatibilities on 1-D and 2-D PAGE. It was concluded that blue silver is slightly better in terms of stain efficiency than Neuhoff CCB, but it presented worse MS compatibility. Neuhoff CCB presented better MS compatibility and superior linearity but worse sensitivity than silver stains. Among the five silvering procedures, He SN showed the best MS compatibility and a reasonable staining efficiency; Yan SN lowered the chances of obtaining the protein identity by PMF but gave the best stain efficiency; Vorum SN gave a very clear background and a great contrast, while Blum SN was the worst in this respect. The implications of these results for the selection of a convenient stain are discussed according to specific objectives as well as practical aspects.     

Live cell surface labeling with fluorescent Ag nanocluster conjugates

DNA-encapsulated silver clusters are readily conjugated to proteins and serve as alternatives to organic dyes and semiconductor quantum dots. Stable and bright on the bulk and single molecule levels, Ag nanocluster fluorescence is readily observed when staining live cell surfaces. Being significantly brighter and more photostable than organics and much smaller than quantum dots with a single point of attachment, these nanomaterials offer promising new approaches for bulk and single molecule biolabeling.     

Dye,fluorophores and pigment coloration of nanofibers produced by electrospinning

Polymeric nanofibers produced by the electrospinning technique are widely used in industrial scale production. Nanofibers chiefly find applications in filtration media and active–barrier surfaces for medical, biological, and military applications. In such applications, the quantity and the uniformity of the nanofibers distribution play a leading role in the product characteristics. For this reason, there is considerable interest regarding the nanofibers recovering quantification and simplification of the qualitative analysis. With the aim to improve and simplify the nanofibers relevability, a coloration approach for nanofibers has been designed and tested. The coloration has been carried out by organic dyes, pigments, and organic fluorophores and the consequent nanofibers' color has been analyzed by optical analysis, colorimetry, and spectroscopy. The coloration obtained by different dyes has been compared and their effect on the nanofibers relevability has been investigated. Moreover, the leading role of the light scattering phenomenon on the nanofiber coloration efficacy has been investigated by comparing the coloration response of nanofibers and film samples on equal terms of dye content. The study has been carried out using polyamide‐6 (PA6) as the testing polymer but the recovering quantification, the coloration approach, and the interaction between light and nanofibers can be extended to all the electrospinnable polymers. Copyright © 2008 John Wiley & Sons, Ltd.     

Green Biosynthesis of Silver Nanoparticles Using ‘Polygonum Hydropiper’ and Study its Catalytic Degradation of Methylene Blue

The green synthesis of silver nanoparticles with the small size and high stability paved the way to improve and protect the environment by decreasing the use of toxic chemicals and eliminating biological risks in biomedical applications. Plant mediated synthesis of silver nanoparticles is gaining more importance owing its simplicity, rapid rate of synthesis of nanoparticles and eco-friendliness. In this study, focus on biosynthesis of silver nanoparticles using Polygonum hydropiper extract and its catalytic degradation of hazardous dye, methylene blue has been highlighted. The rapid reduction of silver (Ag) ions was monitored using UV-Visible spectrophotometer and showed formation of silver nanoparticles within less than one hour with maximum absorption of silver nanoparticles at 430 nm. The major functional groups present in the synthesis responsible for the formation of silver nanoparticles. It was identified by using Fourier Transform Infrared spectrophotometer (FTIR). Field Electron Scanning Microscope (FESEM) was used to characterise the nanoparticles synthesized using P.hydropiper. The morphology of silver nanoparticles was predominantly spherical and aggregated into irregular structure with average diameter of 60 nm. In addition, this report emphasizes the effect of the silver nanoparticles on the degradation rate of hazardous dyes by sodium borohydride (NaBH₄). The efficiency of silver nanoparticles as a promising candidate for the catalysis of organic dyes by NaBH₄ through the electron transfer process is established in the present study.     

Applying Direct Yellow 11 to a modified Simons’ staining assay

For quantification of overall fiber accessibility of lignocellulosic substrates, Direct Yellow 11 (C.I. 40000) is a suitable alternative to the discontinued Pylam Products’ dye Direct Orange 15 (C.I. 40002/40003). In this study we present a side-by-side comparison between the two azo-stilbene dyes. We characterize individual dye fractions and provide equations to determine individual concentrations. We present a modified Simons’ staining protocol incorporating the high molecular weight fraction of Direct Yellow 11. We perform tests on lignin, cellulosic, and lignocellulosic materials. In all tests, the two dyes perform similarly and satisfy many accessibility measurement criteria. We demonstrate that the adsorption of Direct Yellow 11 onto a substrate correlates with that substrate’s propensity for enzymatic hydrolysis. We confirm this correlation on a series of organic solvent pretreatments and on a series of lignocellulosic substrates. Finally, we outline the inherent limitations of performing adsorption experiments with Direct Yellow 11 and other high molecular weight dyes.     

Surface-enhanced raman spectra of dyes and organic acids in silver solutions: chloride ion effect

We have recorded surface-enhanced Raman (SER) spectra of two different classes of compounds, cationic dyes and organic acids, and studied their chloride ion effects on the surface-enhanced Raman scattering (SERS) activities of the silver solution. For the positive charge dyes, rhodamine 6G (R6G) and 1,1'-dimethyl-2,2'-cyanine iodide (DECI), no SERS could be observed without the addition of chloride ions because of lack of the electrostatic interaction between the dye species and the silver particles in the silver solution. The chloride ions served to enlarge silver particles and to contribute the existence of the surface active sites, making the silver solution SERS active to the dye samples. Surface-enhanced resonance Raman scattering (SERRS) intensity of the dye molecules increased with the chloride ion concentration. After reaching a maximum intensity, a Cl- quenching effect on the intensity took place. For the organic acids, benzoic acid and p-aminobenzoic acid (PABA), SERS could be observed without the coexistence of chloride ions. The intensity of the Raman scattering did not vary significantly in the presence of small amount of chloride ion. At high Cl- concentration, quenching SERS intensity began to take effect.     

Determination of hexavalent chromium by on-line dialysis ion chromatography in a matrix of strong colourants and trivalent chromium

Hexavalent chromium detection in the presence of a high load of colourants without any false positive and in-procedure oxidation of Cr(III) is an important area of study. Colourants are a class of interfering substances in many spectroscopic analyses and chromatographic separations and detection. A purification method using an on-line dialysis technique for ion chromatography (IC) has been developed to remove water-soluble anionic dyes and particulate colourants and other substances to facilitate Cr(VI) quantification and the method is discussed. The dialysis was optimized with Cr(VI) standard solutions for quantification. The efficacy of the procedure for the removal of anionic dyes and detection of Cr(VI) was checked with a Cr(VI) spiked synthetic preparation containing a water-soluble dye and trivalent chromium. Soluble Cr(VI) extracted with organic dyes from environmental samples was analyzed. The method has a detection limit of 5 microg/l, recovery rate of 100% and analysis time less than 20 min.     

螺共轭效应及其应用

螺共轭术语是1967年由Simmons和Fukunaga首次提出的, 由于其特殊的立体电子效应而引起了科学家们的广泛关注. 综述了40年来有关螺共轭的理论发展以及在发光材料、光致变色材料、非线性光学材料、螺环染料、有机导体等方面的研究现状, 展望了螺共轭效应的应用前景, 并提出了一些新的设想.     

Synthesis and characterization of surface-enhanced Raman scattering tags with Ag/SiO2 core-shell nanostructures using reverse micelle technology

A novel simplified method for synthesis of surface-enhanced Raman scattering tags has been reported. This synthesis method is based on reverse micelle technique using Igepal CO-520 as a surfactant and the mixed solution of silver nitrate and rhodamine dyes with isothiocyanate group as water pool followed by hydrazine hydrate reduction and TEOS polymerization leading to the formation of silica layer surrounding the silver core. Compared to the method reported in literature, the proposed methodology eliminates the necessity of vitrophilic pretreatment and makes it possible to complete all different processes including the preparation of silver nanoparticles, the conjugation of dye molecules and the formation of silica shell in the microreactor. The nanoparticle-based surface-enhanced Raman tags obtained are composed of silver core conjugated with rhodamine dyes and an encasing silica shell. Both the dyes themselves and the Ag/SiO2 core-shell nanoparticles without the encapsulation of dyes exhibit no Raman signals. However, the Ag/SiO2 core-shell nanoparticles exhibit strong Raman signals when encapsulated with these dyes. This is due to the appearance of fluorescence quenching and surface-enhanced Raman scattering effect resulting from the conjugation of dyes and silver core. The Raman tags were characterized using transmission electron microscopy (TEM), UV-visible absorption spectrometry, and Raman spectrometry.     

Electrocatalytic studies using silver–clay composite — a novel material

A clay-modified electrode (CME) consisting of novel colloidal silver–montmorillonite clay composite material (Ag⁰_n–MM) has been prepared and characterized. The study on its ability to enhance the redox reactions of phenothiazine dyes reveals that the nanosize particle nature of the silver is retained in the film, as reflected from cyclic voltammograms and photogalvanic studies. The photogalvanic current observed for the dyes in this composite material shows an enhanced anodic photocurrent versus the case in ion-exchanged CMEs, where the dyes show a cathodic photocurrent. The unique behavior is explained by a suitable mechanism.     

An optimal method of DNA silver staining in polyacrylamide gels

A silver staining technique has widely been used to detect DNA fragments with high sensitivity on polyacrylamide gels. The conventional procedure of the silver staining is tedious, which takes about 40-60 min and needs five or six kinds of chemicals and four kinds of solutions. Although our previous improved method reduced several steps, it still needed six kinds of chemicals. The objective of this study was to improve further the existing procedures and develop an optimal method for DNA silver staining on polyacrylamide gels. The novel procedure could be completed with only four chemicals and two solutions within 20 min. The steps of ethanol, acetic acid, and nitric acid precession before silver impregnation have been eliminated and the minimal AgNO3 dose has been used in this up-to-date method. The polyacrylamide gel of the DNA silver staining displayed a golden yellow and transparent background with high sensitivity. The minimum 0.44 and 3.5 ng of DNA amount could be detected in denaturing and nondenaturing polyacrylamide gel, respectively. This result indicated that our optimal method can save time and cost, and still keep a high sensitivity for DNA staining in polyacrylamide gels.     

Improved dynamic range of protein quantification in silver‐stained gels by modelling gel images over time

Silver staining is a commonly used protein stain to visualise proteins separated by 2‐DE. Despite this, the technique suffers from a limited dynamic range, making the simultaneous quantification of high‐ and low‐abundant proteins difficult. In this paper we take advantage of the fact that silver staining is not an end‐point stain by photographing the gels during development. This procedure provides information about the change in measured absorbance for each pixel in the protein spots on the gel. The maximum rate of change was found to be correlated with the amount of applied protein, providing a new way of estimating protein amount in 2‐DE gels. We observed an improvement in the dynamic range of silver staining by up to two orders of magnitude.     

Counterion-dye staining for DNA in electrophoresed gels using indoine blue and methyl orange

In this study, we describe a sensitive staining method for DNA in agarose and polyacrylamide gels using organic visible dyes, indoine blue (IB) and methyl orange (MO). The counterion-dye staining method uses two oppositely charged dyes to form a hydrophobic ion pair complex in the staining solution. A decrease in the number of free forms of dyes in staining solution can enhance the selectivity of binding between the dye and DNA, and can reduce nonspecific background staining. As a result, the sensitivity of counterion-dye staining was significantly improved compared with other dye-based staining. This method uses a staining solution consisting of 0.008% IB, 0.002% MO, 10% ethanol and 0.2 M sodium acetate at pH 4.7, and can detect 5 ng of lambda DNA/HindIII within 60 min in agarose gels and 10 ng of PhiX174 DNA/HaeIII within 20 min in polyacrylamide gels.     

短波长菁染料对氯化银乳剂的光谱增感作用

本文利用紫外 可见吸收光谱、光谱曝光等手段研究了5种短波长菁染料,比较了它们在氯化银乳剂上的光谱增感作用,得出3种较好的短波长增感染料,并研究它们在氯化银颗粒上的吸附行为.结果表明,这3种染料在氯化银颗粒上均有吸附,并能有效地提高氯化银乳剂在短波长区的感光度.     

Usefulness of visible dyes for the staining of protein or DNA in electrophoresis

Since 1993, we have studied visible organic dye stains for protein or DNA to improve methodologies and developed the counterion dye staining method. The method employs two oppositely charged dyes that form an ion-pair complex in the staining solution. The selective binding of free dye to protein or DNA in the staining solution improves detection sensitivity and speed. It is a rapid and sensitive procedure, involving fixing/staining or staining/quick destaining steps that are completed in 1-1.5 h. The lowest detection limits achieved are 4-8 ng of protein on polyacrylamide gels and approximately 10 ng of DNA on agarose gels. The focus of this review is to chronicle the development and current status of the counterion dye staining method for detection of protein or DNA. As an extended application of visible dyes, we also discuss the visible dye staining method for detecting protein on blotting membranes developed in our laboratory.     

On the relationship of amino acid composition to silver staining of proteins in electrophoresis gels: II. Peptide sequence analysis.

The quantification of proteins in silver-stained electrophoresis gels has been limited by the differences in "stainability" of different proteins. Despite efforts by many researchers, the precise basis of the reaction between silver reagents and polypeptides is still unclear, and, depending on the formulation, may even differ. We have tested the hypothesis that differences in stainability among proteins can be attributed to differences in di- or tripeptide composition. The results indicate that some order of protein structure other than short peptides accounts for the staining differences observed.