An improved mechanically durable electrophoresis gel matrix that is fully compatible with fluorescence-based protein detection technologies

Unfortunately, conventional large-format polyacrylamide gels are mechanically fragile, often tearing during the subsequent manipulations required for visualization of the proteins. This problem is compounded when large-format two-dimensional gels are subjected to multiple staining procedures in order to detect different classes of proteins, such as total protein, phosphoproteins, and glycoproteins. A mechanically durable liquid polyacrylamide-based matrix has been developed that, upon polymerization, facilitates the handling of one-dimensional and two-dimensional gels. The matrix, referred to as Rhinohide liquid acrylamide, is stable as a refrigerated solution for up to one year, and forms a polymer-reinforced polyacrylamide gel suitable for electrophoresis, upon addition of catalysts. The matrix is superior to previously reported durable gel matrices in that it does not cause distortion of high-molecular-weight bands and does not suffer from other spot morphology artifacts, such as doubling of protein spots in the molecular weight dimension. The matrix is particularly valuable for the analysis of proteins applying multiple applications of fluorescent dyes, as required with serial staining of proteins for phosphorylation, glycosylation, and total protein expression, using Pro-Q Diamond phosphoprotein stain, Pro-Q Emerald glycoprotein stain and SYPRO Ruby protein gel stain, respectively.     

"Liquidless" cell staining by dye diffusion from gels and analysis by laser scanning cytometry: potential application at microgravity conditions in space

BACKGROUND: Conventional staining of cells or tissue sections on microscope slides involves immersing the slides into solutions of dyes then rinsing to remove the unbound dye. There are instances, however, when use of stain solutions is undesirable-e.g., at microgravity conditions in space, where the possibility of accidental spill (many dyes are known carcinogens) introduces health hazard. Likewise, transporting bulk of liquid stains and rinses may be burdensome in certain situations such as field expeditions or combat. METHODS: The "liquidless" staining procedure is proposed in which the dyes are contained in thin strips of hydrated polyacrylamide or gelatin gels that have been presoaked in the stain solutions. Fluorochromes that have affinity to DNA (propidium iodide, PI; 4,6-diamidino-2-phenylindole, DAPI, Hoechst 33342) or to protein (sulforhodamine 101) were used to saturate the gels. The gel strips were placed over the prefixed cells or tissue sections deposited on microscope slides and relatively low (20 g/cm2) pressure was applied to ensure the contact. The cells were also stained by using commercially available mounting media into which DAPI or PI were admixed. Intensity of fluorescence of the PI stained cells was measured by laser scanning cytometry (LSC). RESULTS: Satisfactory cell and tissue staining, with minimal background, was achieved after 10-20 min contact between the cells and gels. Optimal concentrations of the dyes in the solutions used to presoak the gels was found to be 2-4-fold higher than the concentrations used routinely in cytometry. The measurements of intensity of cellular fluorescence by LSC revealed that the staining of DNA was stoichiometric as reflected by the characteristic cellular DNA content frequency histograms with distinct G1, S, and G2/M cell populations and 2:1 ratio of G2/M to G1 peak fluorescence. Individual gels can be saturated with more than a single dye-e.g., to obtain differential DNA and protein staining. Cell staining with DAPI or PI in the gelatin-based mounting media led to high fluorescence background while staining with DAPI in "aqueous" medium was satisfactory. CONCLUSIONS: Relatively fast staining of cells or tissue sections on microscope slides can be achieved by nonconvective dye diffusion using hydrated gels permeated with the dyes, applied to cells at low pressure. The quality of the staining provided by this methodology is comparable to conventional cell staining in dye solutions.     

Fluorography--limitations on its use for the quantitative detection of 3H- and 14C-labeled proteins in polyacrylamide gels

The suitability of fluorography for the detection of 3H- and 14C-labeled proteins on polyacrylamide gradient gels has been investigated. It was found that the absorbance of the fluorographic film image produced by a given level of radioactivity decreased as the acrylamide concentration in the gel increased. The use of Coomassie brilliant blue protein dyes to stain the gel prior to fluorography reduced the absorbance of the fluorographic image. It is concluded that quantitative fluorography can only be applied to unstained gels of a uniform acrylamide concentration.     

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.     

A novel experimental design for comparative two-dimensional gel analysis: two-dimensional difference gel electrophoresis incorporating a pooled internal standard

The comparison of two-dimensional (2-D) gel images from different samples is an established method used to study differences in protein expression. Conventional methods rely on comparing images from at least 2 different gels. Due to the high variation between gels, detection and quantification of protein differences can be problematic. Two-dimensional difference gel electrophoresis (Ettan trade mark DIGE) is an emerging technique for comparative proteomics, which improves the reproducibility and reliability of differential protein expression analysis between samples. In the application of DIGE different samples are labelled with mass and charge matched spectrally resolvable fluorescent dyes and are then separated on the same 2-D gel. Using an Escherichia coli lysate "spiked" with varying amounts of four different known proteins, we have tested a novel experimental design that exploits the sample multiplexing capabilities of DIGE, by including a standard sample in each gel. The standard sample comprises equal amounts of each sample to be compared and was found to improve the accuracy of protein quantification between samples from different gels allowing accurate detection of small differences in protein levels between samples.     

Chain length determination of small double- and single-stranded DNA molecules by polyacrylamide gel electrophoresis.

We describe the use of polyacrylamide gel electrophoresis to estimate chain lengths of double- and single-stranded DNA molecules in the size range 20-1000 base pairs (or nucleotides). Double-stranded DNA molecules of known length produced either by organic synthesis or by restriction endonuclease digestion of viral DNAs were used as standards. The relative electrophoretic mobilities of these standards were examined on both nondenaturing (aqueous) polyacrylamide gels and on denaturing gels containing 7 M urea or 98% formamide. Electrophoretic mobility of DNA is a linear function of the log of molecular weight if appropriate conditions are used, although exceptions are noted. Chain lengths can be conveniently estimated by using as standards bacteriophage gamma DNA restriction fragments or commercially available tracking dyes.     

Using a cationic carbocyanine dye to assess RNA loading in Northern gel analysis.

In Northern blotting, one must have a means of assessing the uniformity of RNA loaded into each lane of a gel. As an alternative to "common gene" controls and previously published nucleic acid dyes (ethidium bromide, acridine orange, methylene blue), we have utilized a cationic carbocyanine dye (Stains All) for the assessment of RNA gel loading uniformity over the range of 5-25 micrograms RNA/lane. The following protocol is suitable for messages of well-characterized mobility and utilizes xylene cyanol as a 4-kb marker; as such, it will migrate between 28S and 18S rRNA over a wide range of agarose concentrations. Optimally, it is best that the message(s) of interest should migrate either as a smaller species than 18S or as a larger species than 28S; this allows either the 28S or 18S ribosomal band to be separated from the message(s) of interest by severing the gel transversely at the xylene cyanol front. Severing the gel in such a manner makes it possible to simultaneously submit that portion of the gel containing either the 28S or 18S rRNA band to Stains All staining while immediately continuing with the transfer of that portion of the gel containing the mRNA of interest. We have found the dye to interact linearly with rRNA whether data were gathered by densitometrically scanning the gels themselves or photographs of the gels.     

The use of a new reactive dye for quantitation of prestained proteins on polyacrylamide gels.

An evaluation of a number of commercial reactive textile dyes with regard to suitability for staining of proteins prior to polyacrylamide gel electrophoresis showed that a blue dye containing a difluorochloropyrimidyl group gave excellent quantitation. The sensitivity was as good as with other dyes suggested for the same purpose. Reaction conditions have been optimized to reach completion in 3 hr at 40°C. A simple device for slicing gels is described. The main errors responsible for experimental scatter are briefly discussed.     

Highly Selective Acridine and Ethidium Staining of Bacterial DNA and RNA

The acridine dyes acridine orange (AO) and coriphosphine O (CPO) and ethidium bromide (EtBr) were used to stain bacterial digests after electrophoresis in native and denaturing (SDS) polyacrylamide gels and were shown to stain DNA and RNA preferentially over other subcellular components in the gels. Vegetative cell digests of Bacillus subtilis, Escherichia coli, Micrococcus luteus, and Staphylococcus aureus showed intense staining of DNA with AO and CPO near the top of the gel, but little or no staining of other cellular constituents. EtBr stained both DNA and RNA in the gels. Protein standards and non-nucleic acid cellular constituents stained faintly with high concentrations (> 100 μM) of AO, lower concentrations (13.9 μM) of CPO, and did not stain with 0.5 μg/ml EtBr in denaturing gels. The complete set of cellular biochemicals was visualized by silver staining, while the protein subset was detected by Coomassie blue staining. The highest concentrations of AO (120 μM) and CPO (13.9 μM) were shown to detect purified DNA in gels with a sensitivity in the range of 25–50 ng per band. This work demonstrates the specificity of acridine and ethidium dyes for nucleic acids, while illustrating the level of non-nucleic acid-specific interactions with other cellular components by staining of electrophoretically separated cellular components in a gel matrix.     

Agar Gel Electrophoresis: an Advantageous Technique for the Investigation of Certain Biological Stains

The agar gel method can be used to study direct dyes, for which paper can not be used because such dyes have a high affinity for the paper. A 1% gel made up with a buffer in the range of pH 9-4, of ionic strength 0.05, and spread on 8 × 10 cm lantern slides provides suitable conditions. Dyes to be tested are placed in 1.5 mm wells made in the agar and subjected to a current of 2.5 ma/cm width, at a potential of about 115 v. Separations, if any, occur in about 20 min. Mobility is affected by ionic strength; values above 0.05 may be less satisfactory by reducing mobility and allowing excessive diffusion of the dye. The method allows resolution of dyes whose molecular charges differ by only one unit. Photographic recording is sharp, since the gel is transparent. The method can be recommended as generally useful for studying both acid and direct dyes.     

Rheological properties and mechanical stability of new gel-entrapment systems applied in bioreactors

The mechanical stability of gels applied for entrapment and retention of biocatalysts in bioreactors is of crucial importance for successful scale-up applications. Gel abrasion in agitated reactors will depend on liquid shear, bubble shear, and wall shear, as well as collisions between the gel particles. As a simplified standardized model system, abrasion of gel beads was studied in 1-m-high bubble columns with controlled aeration, and quantified by measuring the loss of gel material into solution. Gel beads were also taken out to measure stress-strain response during controlled compression. More general rheological properties of different gels were studied by applying a variety of regimes of controlled compression of standardized gel cylinders: Gel strength was measured by recording the fracture properties and the Young's modulus. Viscoelastic properties were revealed by recording creep during compression as well as recovery after compression. Oscillation tests up to 1000 cyclic compressions were applied to compare the fatigue of different gels. Results obtained for Ca-alginate gels, gels of chemically modified polyvinyl alcohol with stilbazolium groups (PVA-SbQ) as well as mixed gels of Ca-alginate and PVA-SbQ are compared with previously published data for kappa-carrageenan, agar, and polyethylene glycol (PEG) gels. It is concluded that material fatigue rather than mechanical properties such as stiffness or fracture stress should be considered when selecting a suitable gel material on the basis of abrasion resistance. The very soft and superelastic PVA-SbQ gel showed no significant fatigue in mechanical tests and no abrasion was detected in the standardized model system used. Ca-alginate gels, however, showed severe irreversible changes due to fatigue at oscillating loads and creep at constant load. Due to their similarities with kappa-carrageenan gels in mechanical tests, it is likely that Ca-alginate would also be sensitive to abrasion. Mixed gels of Ca-alginate and PVA-SbQ represent a complex system with intermediate properties, showing significant fatigue and creep, but elastic properties from the PVA-SbQ gel make it less sensitive than the pure Ca-alginate gel.     

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.     

A vertical submarine electrophoresis apparatus for polyacrylamide minigels

A vertical submarine electrophoresis apparatus for use with minislab polyacrylamide gels is described. The design allows polyacrylamide gels to be run with the same ease and convenience that agarose gels are run with horizontal submarine apparatuses. The vertical submarine features a single buffer chamber with a restriction between the upper and the lower portions of the chamber. Acrylamide gels, cast between 9 X 10-cm glass slides, are inserted into the restriction and are completely immersed in buffer. Thus, current flows primarily through the gel itself, but some current flows through the buffer in the restriction surrounding the gel. Because water-tight separation of buffer chambers is not necessary, time-consuming and/or expensive procedures such as sealing with agarose or using fragile notched glass plates are eliminated. The apparatus can be set up to run a gel in less than 30 s. It is versatile in that gels of varying thickness (0.5, 0.8, 1.5, and 3 mm) can be run on a single apparatus. The apparatus has been used for sodium dodecyl sulfate gels, low ionic strength native gels for nucleoprotein complexes, and composite acrylamide-agarose gels.     

A method for horizontal polyacrylamide slab gel electrophoresis

We present a simplified method of preparation of polyacrylamide gels which is totally analogous to the procedure now widely used to pour and run horizontal agarose gels. The acrylamide is poured into an open air gel mold consisting of a glass plate with a masking tape border and a comb. It is subsequently run in a submarine horizontal electrophoresis apparatus. The electrophoretic mobility and resolution of DNA fragments obtained in such gels are identical to results obtained with gels poured and run in the vertical configuration. Numerous advantages of horizontal polyacrylamide gel electrophoresis are discussed.     

Two-dimensional fluorescence difference gel electrophoresis for comparative proteomics profiling

Quantitative proteomics is the workhorse of the modern proteomics initiative. The gel-based and MuDPIT approaches have facilitated vital advances in the measurement of protein expression alterations in normal and disease phenotypic states. The methodological advance in two-dimensional gel electrophoresis (2DGE) has been the multiplexing fluorescent two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). 2D-DIGE is based on direct labeling of lysine groups on proteins with cyanine CyDye DIGE Fluor minimal dyes before isoelectric focusing, enabling the labeling of 2-3 samples with different dyes and electrophoresis of all the samples on the same 2D gel. This capability minimizes spot pattern variability and the number of gels in an experiment while providing simple, accurate and reproducible spot matching. This protocol can be completed in 3-5 weeks depending on the sample size of the experiment and the level of expertise of the investigator.     

从琼脂糖凝胶中高效回收DNA技术的探讨

用两只离心管制成的凝胶过滤装置,从电泳后的琼脂糖凝胶中回收DNA片段的简易方法。它依次包括以下步骤：凝胶过滤装置的制作、凝胶切割、凝胶低温冷冻、低温高速离心、ddH20洗胶、DNA纯化和回收效果检测等。用此方法回收的DNA片段产率高、质量纯,可直接用于分子生物学实验的后续操作,如载体连接、PCR模板获得、DNA探针制备、基因测序等。其优点是：DNA片段的回收率高（90％以上）,质量好;操作简便,耗时短;回收装置简单,成本低廉,可进行商品化开发。     

Previsible silver staining of protein in electrophoresis gels with mass spectrometry compatibility

A convenient silver staining method for protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels is described. The method is previsible, sensitive, and mass spectrometry (MS) compatible. Two visible counter ion dyes, ethyl violet (EV) and zincon (ZC), were used in the first staining solution with a detection limit of 2 to 8 ng/band in approximately 1 h. The dye-stained gel can be further stained by silver staining, which is based on acidic silver staining employing ZC with sodium thiosulfate as silver ion sensitizers. Especially, ZC has silver ion reducing power by cleavage of the diazo bond of the dye during silver reduction. The second silver staining can be completed in approximately 1 h with a detection limit of 0.2 ng/band.     

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.     

Electrophoresis of proteins and DNA on horizontal sodium dodecyl sulfate polyacrylamide gels

An inexpensive Plexiglas apparatus which allows a simple and rapid preparation of horizontal polyacrylamide gels of different dimensions for different purposes, is described. Preparation of such gels is as easy and rapid as agarose gel preparation, and polymerized polyacrylamide gels are used to fractionate proteins or small DNA fragments using a common horizontal electrophoretic tank. This apparatus was used to electrophoretically fractionate proteins or DNA for immuno-blot analyses, particularirly in the study of the allergenic response to Parietaria judaica pollen in senescence, for Southern-blot hybridizations and in the study of DNA polymorphisms.     

Interaction of cyanine dyes with nucleic acids: XXXI. Using of polymethine cyanine dyes for the visualization of DNA in agarose gels

Fifteen polymethine cyanine dyes were studied as fluorescent stains for DNA in electrophoretic gels. Among studied cyanines, two dyes CPent V and CCyan 2-O most effectively visualized covalently closed and linear double-stranded DNA molecules in gels under standard conditions using UV-illumination, green filter and black-and-white photo film. Ethidium bromide was 1.2-1.6 times more effective as compared to cyanine dyes in staining of DNA in the concentration range of 8-18 ng, while studied cyanines were more sensitive to DNA quantity above 50 ng.