Cyanine dyes for the detection of double stranded DNA

Twenty three novel cyanine dyes have been applied as fluorescent stains for the detection of nucleic acids in agarose gel electrophoresis. Significant fluorescence enhancement of these dyes in the presence of double stranded DNA was observed. Five dyes offered superior sensitivity in the detection and quantification of DNA, over Ethidium Bromide, the most commonly used nucleic acid stain.     

Protein Detection Methods in Proteomics Research

In proteomics research chemical as well as physical methods are used to detect proteins subsequently to their separation. Physical methods are mostly applied after chromatography. They are either based on spectroscopy like light absorption at certain wavelengths or mass determination of peptides and their fragments with mass spectrometry. Chemical methods are used after two-dimensional electrophoresis and employ staining with organic dyes, metal chelates, fluorescent dyes, complexing with silver, or pre-labeling with fluorophores. In some cases autoradiography is still used. Since all of these techniques are very different in terms of sensitivity, their usefulness for quantitative determinations varies significantly. This review will describe the various protein detection methods applied to electrophoresis gels.     

Analysis of ultra acidic proteins by the use of anodic acidic gels

Ultra acidic proteins, generated by posttranslational modifications, are becoming increasingly important due to recent evidence showing their function as regulatory elements or as intermediates in degradation pathways in bacteria. Such proteins are important in neurodegenerative diseases and embryonic development, and they include the Alzheimer-related tau (τ) protein (resulting from posttranslational modifications) and the phosphor-storage embryonic proteins. The ultra acidic proteins are difficult to study because standard two-dimensional gel electrophoresis is inadequate for their analysis. Here we describe a novel electrophoresis system of anodic acidic gels that can replace isoelectric focusing as the first dimension of separation in two-dimensional electrophoresis. The system is based on a sodium acetate buffer (pH 4.6), is compatible with traditional stains (e.g., Coomassie blue) as well as novel fluorescent dyes (e.g., Pro-Q Diamond), and is quantitative for the analysis of ultra acidic proteins. The anodic acidic gels were used for the functional classification of the ultra acidic part of the Bacillus subtilis proteome, showing significant improvement over traditional two-dimensional electrophoresis.     

Evaluation of saturation labelling two-dimensional difference gel electrophoresis fluorescent dyes

Two-dimensional difference gel electrophoresis (2-D DIGE) enables an increased confidence in detection of protein differences. However, due to the nature of the minimal labelling where only approximately 5% of a given protein is labelled, spots cannot be directly excised for mass spectrometry (MS) analysis and detection sensitivity could be further enhanced. Amersham Biosciences have developed a second set of CyDye DIGE Cy 3 and Cy5 dyes, which aim to overcome these limitations through saturation-labelling of cysteine residues. The dyes were evaluated in relation to their sensitivity and dynamic range, their useability as multiplexing reagents and the possibility of direct spot picking from saturation-labelled gels for MS analysis. The saturation-labelling dyes were superior in sensitivity to their minimal-labelling counterparts, silver stain and Sypro Ruby, however, the resulting 2-D spot pattern was significantly altered from that of unlabelled or minimal-labelled protein. The dyes were found to be useful as multiplexing reagents although preferential labelling of proteins with one dye over another was observed but was controlled for through experimental design. Protein identities were successfully obtained from material directly excised from saturation-labelled gels eliminating the need for post-stained preparative gels.     

Rapid two-dimensional analysis of proteins by ultra-thin layer gel electrophoresis

Identification of qualitative and/or quantitative protein expression differences as well as characterization of specific cell proteomes would further advance molecular cell biology research. Today, one of the most commonly used tools for proteome analysis is two-dimensional gel electrophoresis. Although this technology is informative, it is extremely cumbersome, time-consuming and lacks automation and proper reproducibility. In this paper, we propose an automated separation/detection system capable of rapid two-dimensional analysis of proteins by ultra-thin layer gel electrophoresis with real time imaging of the separated components, using fiber optics based laser induced fluorescence technology. The approach is based on electric field mediated separation in capillary dimensions, along with noncovalent, "in migratio" fluorescent staining methodology. The advantage of the technology discussed over existing techniques is its simplicity, speed and good detection sensitivity.     

Detection technologies in proteome analysis

Common strategies employed for general protein detection include organic dye, silver stain, radiolabeling, reverse stain, fluorescent stain, chemiluminescent stain and mass spectrometry-based approaches. Fluorescence-based protein detection methods have recently surpassed conventional technologies such as colloidal Coomassie blue and silver staining in terms of quantitative accuracy, detection sensitivity, and compatibility with modern downstream protein identification and characterization procedures, such as mass spectrometry. Additionally, specific detection methods suitable for revealing protein post-translational modifications have been devised over the years. These include methods for the detection of glycoproteins, phosphoproteins, proteolytic modifications, S-nitrosylation, arginine methylation and ADP-ribosylation. Methods for the detection of a range of reporter enzymes and epitope tags are now available as well, including those for visualizing beta-glucuronidase, beta-galactosidase, oligohistidine tags and green fluorescent protein. Fluorescence-based and mass spectrometry-based methodologies are just beginning to offer unparalleled new capabilities in the field of proteomics through the performance of multiplexed quantitative analysis. The primary objective of differential display proteomics is to increase the information content and throughput of proteomics studies through multiplexed analysis. Currently, three principal approaches to differential display proteomics are being actively pursued, difference gel electrophoresis (DIGE), multiplexed proteomics (MP) and isotope-coded affinity tagging (ICAT). New multiplexing capabilities should greatly enhance the applicability of the two-dimensional gel electrophoresis technique with respect to addressing fundamental questions related to proteome-wide changes in protein expression and post-translational modification.     

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.     

Protein stains for proteomic applications: which, when, why?

This review recollects literature data on sensitivity and dynamic range for the most commonly used colorimetric and fluorescent dyes for general protein staining, and summarizes procedures for the most common PTM-specific detection methods. It also compiles some important points to be considered in imaging and evaluation. In addition to theoretical considerations, examples are provided to illustrate differential staining of specific proteins with different detection methods. This includes a large body of original data on the comparative evaluation of several pre- and post-electrophoresis stains used in parallel on a single specimen, horse serum run in 2-DE (IPG-DALT). A number of proteins/protein spots are found to be over- or under-revealed with some of the staining procedures.     

Development of improved cell lysis, solubilization and imaging approaches for proteomic analyses

Analysis of complex biochemical processes at the level of the proteome requires methods that quantitatively solubilize cytosolic and membrane bound proteins yet are compatible with isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition, it is often necessary to employ several highly sensitive detection methods to identify key proteins that are modified or exhibit a change in expression levels in response to a given experimental stimulus or condition. Methods were developed that efficiently extract tissues or lyse cultured cells and quantitatively solubilize proteins in a single step without the need to shear nucleic acids. These approaches utilize urea, thiourea, a mixture of detergents, low levels of an ampholyte blend, reductant and a combination of alcohols. To aid in the detection of low abundance proteins and the accurate identification of specific proteins of interest in these samples, two approaches were pursued. In one, proteins are transferred from two-dimensional (2-D) gels to blot membranes. Proteins are then detected by staining with SYPRO Ruby and the resulting 2-D protein pattern is captured using a charge-coupled device (CCD) camera. The blots are then probed with antibodies directed against the protein(s) or functionalities of interest. The resulting chemiluminescent blot image is also generated with the CCD camera and the fluorescent SYPRO Ruby image is recaptured again without moving the membrane. It is thereby possible to generate a direct image overlay of the blot pattern on that of the stained protein pattern. This approach significantly aids in the accurate identification of the dye-stained protein that is detected by the specific antibody. In addition to detecting protein post-gel transfer, a second approach utilizes protein samples labeled with fluorescent dyes prior to 2-D electrophoresis in an effort to increase the sensitivity of protein detection and to facilitate protein quantitation. It is also possible to stain the blots with different dyes and overlay these images as well. Using these approaches, it is possible to perform more rapid and accurate comparative analyses and proteomic, post-gel characterization of proteins of interest than using comparative image analysis of multiple gels.     

Principles and methods for the analysis and purification of synthetic deoxyribonucleotides by high-performance liquid chromatography

The need for high-purity oligodeoxyribonucleotides for various applications has resulted in the development of novel synthesis, purification, and analytical techniques, A diversity of methods, including polyacrylamide slab gel electrophoresis, capillary gel electrophoresis, as well as high-performance liquid chromatography (HPLC), have been successfully used to aid in the characterization and isolation of these synthetic compounds. The information contained in this review article primarily details both the theoretical and practical aspects related to the use of HPLC for the analysis and purfication of synthetic DNA. In addition, a variety of postsynthesis sample preparation protocols, commonly employed prior to and after HPLC, are described.     

Characterization of six textile dyes as fluorescent stains for flow cytometry.

Few fluorescent stains specific for cell constituents other than DNA are available. To assess their potential use as fluorescent stains for flow cytometry, the cell staining specificity of 55 compounds, originally synthesized for use as textile dyes and fluorescent brighteners, was explored and their excitation and emission wavebands determined. From these, six dyes were chosen for more detailed analysis. All six are vital stains, with excitation wavelengths allowing their use with an argon ion laser, and specific for a range of cell structures including mitochondria, Golgi bodies, lipid droplets, nuclear membrane, and endoplasmic reticulum. Concentrations as low as 0.01-0.25 microM were found to be adequate for most purposes, and high background fluorescence was not a problem. Their specificity allows differentiation between non-cycling and cycling cells. The properties of two of the stains allows their combination with propidium iodide or ethidium bromide for simultaneous determination of DNA content profiles. Being vital stains, usable at very low concentrations, and specific for a range of cell organelles, these six stains may be of considerable utility in flow cytofluorometry. We suggest that other textile dyes may be of use in flow cytofluorometry, or that their structures may form a starting point for the synthesis of further fluorescent stains of enhanced specificity.     

Identification of Fusarium graminearum in cereal samples by DNA Detection Test Strips

AIMS: Development of a fast, sensitive and easy to handle method for the detection of Fusarium graminearum contamination in cereal samples by PCR. METHODS AND RESULTS: DNA Detection Test Strips were used for PCR-product detection and the method was compared to agarose gel electrophoresis. A minimum of 0.26 ng of purified target DNA was detectable with the Test Strip Detection limit in less contaminated samples was slightly lower when gel electrophoresis was used for amplicon detection. In highly contaminated samples, detection limits of both methods were similar. CONCLUSIONS: Detection of PCR products was performed in 20 min without the need of special technical equipment or hazardous fluorescent DNA dyes. SIGNIFICANCE AND IMPACT OF THE STUDY: The new method described is useful for the screening of cereals in industrial quality control.     

Improved mass spectrometry compatibility is afforded by ammoniacal silver staining

Sequence coverage in MS analysis of protein digestion-derived peptides is a key issue for detailed characterization of proteins or identification at low quantities. In gel-based proteomics studies, the sequence coverage greatly depends on the protein detection method. It is shown here that ammoniacal silver detection methods offer improved sequence coverage over standard silver nitrate methods, while keeping the high sensitivity of silver staining. With the development of 2D-PAGE-based proteomics, another burden is placed on the detection methods used for protein detection on 2-D-gels. Besides the classical requirements of linearity, sensitivity, and homogeneity from one protein to another, detection methods must now take into account another aspect, namely their compatibility with MS. This compatibility is evidenced by two different and complementary aspects, which are (i) the absence of adducts and artefactual modifications on the peptides obtained after protease digestion of a protein detected and digested in - gel, and (ii) the quantitative yield of peptides recovered after digestion and analyzed by the mass spectrometer. While this quantitative yield is not very important per se, it is however a crucial parameter as it strongly influences the S/N of the mass spectrum and thus the number of peptides that can be detected from a given protein input, especially at low protein amounts. This influences in turn the sequence coverage and thus the detail of the analysis provided by the mass spectrometer.     

Use of polynucleotide/polyacrylamide-gel electrophoresis as a sensitive technique for the detection and comparison of ribonuclease activities.

A technique is described in which the incorporation of a polynucleotide substrate into the matrix of a polyacrylamide gel allows the use of electrophoresis for the detection of polycationic ribonuclease activity rather than simply the presence of protein. Because use is made of the catalytic properties of ribonucleases, polynucleotide/polyacrylamide-gel electrophoresis is apparoximately 10(5) times more sensitive for the detection of these enzymes than conventional gel electrophoresis with the use of protein-staining dyes. Initial studies showed that the poor migration, in the gels, of highly charged polycationic ribonucleases in the presence of negatively charged synthetic polynucleotides could be overcome by high concentrations of spermine. The positively charged polyamine, by neutralizing the polyanionic polynucleotide, enabled these basic enzymes to migrate considerable distances in the gel. Electrophoresis of the RNAases under conditions of low pH, and incubation of the gel at neutral pH followed by staining for polynucleotide, resulted in coloured gels containing clear bands that define regions of enzyme activity. Alterations in spermine concentration or substrate identity caused changes in the positions of these bands, suggesting a dynamic interaction among the enzyme, polyamine and polynucleotide. Because of the advantages, in terms of selectivity and sensitivity of polynucleotide/polyacrylamide-gel electrophoresis, this technique was used to demonstrate the nuclease homogenity of three purified bovine muscle enzymes, and to compare these enzymes with each other, as well as with bovine pancreatic ribonuclease A.     

Sensitive, quantitative, and fast modifications for Coomassie Blue staining of polyacrylamide gels

In spite of the high sensitivity of silver staining and the wide dynamic range of various fluorescent detection methods, Coomassie Brilliant Blue staining is still the most widely used protein detection technique for proteins separated by polyacrylamide gel electrophoresis. There are several reasons: Low price, Visible with the eye, Desk top scanners can be employed for image acquisition, Better for quantitative analysis than silver staining, Possible modifications for fast or highly sensitive staining, Mass spectrometry compatible.     

A comparison of rapid electrophoretic and chromatographic methods for the investigation of common histological dyes

Summary The compositions of fifty-nine common histological dyes, as well as duplicate samples of several dyes from different suppliers, have been studied by agar gel electrophoresis, agarose gel electrophoresis, paper electrophoresis, paper chromatography and thin layer chromatography. Tables are presented to show the number of components present in each dye as disclosed by the different methods; the cases where duplicate samples were available are summarised in a separate table.On the basis of effectiveness and convenience agar gel electrophoresis and thin layer chromatography were by far the best methods. The Chromatographic method was of slightly wider applicability but as electrophoretic methods gave information on dye charge, agar gel electrophoresis was the best single method.     

New amino and acetamido monomethine cyanine dyes for the detection of DNA in agarose gels

Some new monomethine cyanine dyes derived from quinoline and benzothiazole have been prepared and characterized by (1)H and (13)C NMR, FTIR, FABHRMS, and visible spectroscopy. The dyes containing amino and acetamido groups were conveniently synthesized by the condensation of two p-toluenesulfonate heterocyclic quaternary salts and were obtained in the forms of iodide, bromide, and tosylate counteranions. These dyes were compared to ethidium bromide as stains for DNA in electrophoretic gels. The overall results obtained for the sensitivity of these dyes suggest the suitability of acetamido moiety over the amine one and bromide as the counteranion when compared with iodide and tosylate, with a similar capacity of DNA detection in relation to the ethidium bromide stain over the concentration range of 1-3ng.     

Capillary affinity gel electrophoresis

Capillary affinity gel electrophoresis is a new technique for the recognition of the specific DNA base and/or sequence. This technology is also applicable to the characterization of binding properties of DNA-based drugs, chiral separation, and the selective separation of antibody mimetics using imprinted polymers. This article reviews the present state of studies on the capillary affinity gel electrophoresis, including the principle, theory, methods, and applications of this technology. The great potential of capillary affinity gel electrophoresis for the detection of the mutation onDNA is illustrated.     

A quantitative method for detection of spliced X-box binding protein-1 (XBP1) mRNA as a measure of endoplasmic reticulum (ER) stress

Endoplasmic reticulum (ER) stress is increasingly recognized as an important mechanism in a wide range of diseases including cystic fibrosis, alpha-1 antitrypsin deficiency, Parkinson's and Alzheimer's disease. Therefore, there is an increased need for reliable and quantitative markers for detection of ER stress in human tissues and cells. Accumulation of unfolded or misfolded proteins in the endoplasmic reticulum can cause ER stress, which leads to the activation of the unfolded protein response (UPR). UPR signaling involves splicing of X-box binding protein-1 (XBP1) mRNA, which is frequently used as a marker for ER stress. In most studies, the splicing of the XBP1 mRNA is visualized by gel electrophoresis which is laborious and difficult to quantify. In the present study, we have developed and validated a quantitative real-time RT-PCR method to detect the spliced form of XBP1 mRNA.     

Specificity and Sensitivity of Insulin Staining by Aldehyde Fuchsin, Pseudoisocyanin and Toluidine Blue

Aldehyde fuchsin, pseudoisocyanin and toluidine blue, histochemical dyes reported to be specific for insulin-containing granules of the pancreatic beta cell, were applied to insulin fixed in polyacrylamide gel by disc electrophoresis. Two major and four minor bands were resolved as demonstrated by staining with amidoschwarz; only the two major bands, were stained by aldehyde fuchsin. The addition of serum did not affect this reaction. Serum or insulin components gave no metachromatic reactions to the other stains. Under the conditions applied, aldehyde fuchsin is the only one of these dyes specific for insulin in this, system, but this stain is not sufficiently sensitive to detect normal serum levels of the hormone.