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PubMed2002

Cross-linked polyacrylamide gel electrophoresis of single-stranded DNA for microfabricated genomic analysis systems.

Microfabricated devices are poised to offer inexpensive self-contained alternatives to conventional benchtop-scale laboratory equipment for performing a variety of important DNA analysis assays. In order to realize the dramatic cost savings possible through photolithographic fabrication techniques, these devices must occupy an extremely compact footprint on the silicon wafer. This requirement implies that electrophoretic separations must be performed over ultrashort distances. Employing cross-linked polyacrylamide gels in place of conventional uncross-linked sieving media offers a convenient strategy to achieve this goal. In this paper, we show how the increased resolving power offered by cross-linked polyacrylamide gels, along with improved sample injection techniques, can be exploited to enhance separation performance in microscale systems. We use these techniques to perform high-resolution gel electrophoresis of single-stranded DNA fragments in microfabricated devices over separation distances of 1.5 cm or less. The results presented here are in agreement with theoretical predictions and suggest that it is possible to perform DNA sequencing on compact microchips. More importantly, the separation performance demonstrated in this work is already more than adequate to perform a number of important genomic assays imposing less stringent resolution requirements than sequencing. Successfully adapting even a few of these assays to the microdevice format has the potential to provide a new generation of inexpensive and portable devices suitable for direct end-user applications.

Acrylic Resins↗
PubMed2002

DNA sequencing of close to 1000 bases in 40 minutes by capillary electrophoresis using dimethyl sulfoxide and urea as denaturants in replaceable linear polyacrylamide solutions.

The goal of this work was to reduce the capillary electrophoresis (CE) separation time of DNA sequencing fragments with linear polyacrylamide solutions while maintaining the previously achieved long read lengths of 1000 bases. Separation speed can be increased while maintaining long read lengths by reducing the separation matrix viscosity and/or raising the column temperature. As urea is a major contributor to the separation buffer viscosity, reducing its concentration is desirable both for increase in the separation speed and easier solution replacement from the capillary. However, at urea concentrations below 6 M, the denaturing capacity of the separation buffer is not sufficient for accurate base-calling. To restore the denaturing properties of the buffer, a small amount of an organic solvent was added to the formulation. We found that a mixture of 2 M urea with 5% v/w of dimethyl sulfoxide (DMSO) resulted in 975 bases being sequenced at 70 degrees C in 40 min with 98.5% accuracy. To achieve this result, the software was modified to perform base-calling at a peak resolution as low as 0.24. It is also demonstrated that the products of thermal decomposition of urea had a deleterious effect on the separation performance at temperatures above 70 degrees C. With total replacement of urea with DMSO, at a concentration of 5% v/w in the same linear polyacrylamide (LPA)-containing buffer, it was possible to increase the column temperature up to 90 degrees C. At this temperature, up to 951 bases with 98.5% accuracy could be read in only 32 min of separation. However, with DMSO alone, some groups of C-terminated peaks remained compressed, and column temperature at this level cannot at present be utilized with existing commercial instrumentation.

Acrylic Resins↗
PubMed2001

Identification and mapping of human saphenous vein medial smooth muscle proteins by two-dimensional polyacrylamide gel electrophoresis.

Changing smooth muscle phenotype and abnormal cell proliferation are important features of vascular pathology, including the failure of saphenous vein bypass grafts. We have characterised and mapped protein expression in human saphenous vein medial smooth muscle, using two-dimensional (2-D) polyacrylamide gel electrophoresis. The 2-D system comprised a nonlinear immobilised pH 3-10 gradient in the first dimension (separating proteins with isoelectric point values between pH 3-10), and 12%T total gel concentration sodium dodecyl sulphate polyacrylamide gel electrophoresis in the second dimension (separating proteins in the range 14,000-200,000 Daltons). Using a combination of peptide mass fingerprinting by matrix-assisted laser desorption/ionisation-time of flight mass spectrometry and partial amino acid sequencing by nanospray tandem mass spectrometry, a subset of 149 protein spots was analysed, with 129 protein spots being identified and mapped. The data presented here are an important addition to the limited knowledge of venous medial smooth muscle protein expression in vivo. Our protein map will facilitate the identification of proteins differentially expressed in human saphenous vein bypass grafts. In turn, this may lead to the elucidation of molecular events involved in saphenous vein bypass graft failure. The map should also provide a basis for comparative studies of protein expression in vascular smooth muscle of varying origins.

Electrophoresis, Gel, Two-Dimensional↗
PubMed1989

Determination of total and free concentration of propranolol in human plasma by displacement electrophoresis in a two-layer polyacrylamide gel using fluorimetric detection.

A new method based on displacement electrophoresis has been developed for the determination of the total and free concentration of propranolol, a beta-adrenergic blocker drug, in plasma. To determine the total concentration the drug is extracted from human plasma into a chloroform + heptane mixture in the presence of ammonia. After evaporation of the solvent mixture the residue is submitted to displacement electrophoresis in a glass tube containing a two-layer polyacrylamide gel. When the propranolol electrophoretically leaves the gel column it is transferred by a buffer flow to the cuvette of a fluorimeter for continuous detection and quantification. The concentration of the free non-protein bound drug can be determined by the same displacement electrophoresis technique following extraction of the plasma sample into the above organic solvent mixture in the absence of ammonia. Alternatively the extraction procedure can be exchanged for dialysis for 1 h. To decrease the risk that large size material might comigrate with propranolol two layers of polyacrylamide are used, one having small pores to retard such material. In addition, we use fluorimetric detection which means that many possible contaminants are not recorded and therefore do not affect a quantitative determination of propranolol.

Ammonia↗
PubMed1988

Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250.

An improved procedure for staining of proteins following separation in polyacrylamide gels is described which utilizes the colloidal properties of Coomassie Brilliant Blue G-250 and R-250. The new method is based on addition of 20% v/v methanol and higher concentrations of ammonium sulfate to the staining solution previously described. The method combines the advantage of much shorter staining time with high sensitivity, a clear background not requiring destaining, stepwise staining, and stable fixation after staining. The method has been applied to staining of polyacrylamide gels after sodium dodecyl sulfate-electrophoresis and isoelectric focusing in carrier ampholyte-generated pH gradients.

Acrylic Resins↗
PubMed1989

Characterization of human residual catalase of an acatalasemic patient by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by electrophoretic blotting and immunodetection.

Isoelectric points and subunit sizes of catalases in human blood and human cultured skin fibroblasts from acatalasemic and normal subjects were analyzed by isoelectric focusing in agarose gel and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively, followed by electroblotting to polyvinylidene difluoride membranes for immunodetection. The results indicated that the isoelectric point of residual catalase in the C fraction prepared from acatalasemic erythrocytes was identical with that of catalase prepared from normal erythrocytes. The residual catalase in homogenates of acatalasemic cultured skin fibroblasts also reacted with anticatalase rabbit serum and had an isoelectric point identical with that of normal catalase. Subunit sizes of normal and acatalasemic catalases in the C fractions of erythrocytes were also found to be identical on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by electroblotting and immunoenzymatic amplification. The results indicated no substantial difference in molecular size and charge of catalase proteins between normal and acatalasemic erythrocytes and fibroblasts.

Acatalasia↗
PubMed1989

Fixation of amino acids and oligopeptides by freeze-drying in polyacrylamide gel electrophoresis.

Following polyacrylamide gel electrophoresis in an acetic/formic acid buffer, pH 2.0, in fabric reinforced gels, amino acids and oligopeptides could successfully be fixed in the gel by freeze-drying. Lyophilization of the fabric reinforced polyacrylamide gel after electrophoresis resulted in a dry film which absorbed ninhydrin solution quickly and uniformly, thus improving the detection limit for amino acids and oligopeptides with molecular weights ranging from 189-1045. Most amino acids were detected with a sensitivity of 0.1-0.25 microgram and for oligopeptides the detection limit was found to be 0.5-5 microgram.

Amino Acids↗
PubMed1989

Detection of beta-1,3-glucanase activity after native polyacrylamide gel electrophoresis: application to tobacco pathogenesis-related proteins.

Beta-1,3-Glucanase (laminarinase) activity was detected after polyacrylamide gel electrophoresis under native conditions by using laminarin as substrate. Following incubation of gels, laminarin was stained with Aniline Blue. Under UV illumination, lysis zones appeared as dark bands against a fluorescent background. As low as 0.001 unit of commercial Penicillium laminarinase could be observed after incubating the polyacrylamide gel for 45 min at pH 5.0. Extracts of commercial Penicillium laminarinase exhibited four bands with lytic activity towards laminarin. Analysis of intercellular fluid extracts of tobacco mosaic virus-infected tobacco leaves revealed four beta-1,3-glucanases corresponding to three acidic pathogenesis-related proteins, b4 (2), b5 (N) and b6b (0), and one basic protein. The presence of laminarin in gels retarded the migration of some proteins with beta 1,3-glucanase activity. This change in electrophoretic mobility could be used as a complementary affinity test for identifying proteins with beta-1,3-glucanase activity.

Electrophoresis, Polyacrylamide Gel↗
PubMed1990

Isolation and recovery of acidic oligosaccharides from polyacrylamide gels by semi-dry electrotransfer.

Acidic oligosaccharides derived from glycosaminoglycan heparin were separated by polyacrylamide gradient gel electrophoresis (PAGE). The gel could be visualized using Alcian Blue dye to give a pattern of highly resolved, well defined bands. The particular banding pattern obtained was the result of a heparinase catalyzed depolymerization which afforded oligosaccharide products that differed in size by one disaccharide unit. The separated oligosaccharides could be recovered prior to staining by electroelution onto a positively charged nylon membrane by a semi-dry transfer procedure. Subsequent elution and quantitative recovery of individual oligosaccharides from the membrane was achieved. By using multiple membrane layers a second separation dimension was obtained, resulting in increased oligosaccharide purity proportional to transfer depth. Preparative gradient polyacrylamide gel electrophoresis followed by semi-dry electro-transfer and recovery represents a novel method for the preparation of homogeneous acidic oligosaccharides.

Alcian Blue↗
PubMed1990

Sequence analysis of proteins separated by polyacrylamide gel electrophoresis: towards an integrated protein database.

Improved technologies or the synergistic use of complementary methods enhance the efficiency of research and permit the exploration of new approaches for the investigation of complex problems. High sensitivity protein sequence analysis and polyacrylamide gel electrophoresis are such complementary methods. Here we summarize the current status of high sensitivity sequence analysis of proteins separated in polyacrylamide gels and discuss strategies by which this technology can enhance biological research by generating new approaches for the solution of complex, multifacetted problems. Finally, we outline imminent technological advances in the area of high sensitivity protein sequence analysis and argue that further technological developments will ultimately lead to the generation of an integrated protein database (containing structural and functional as well as physiological information in an easily accessible form) of all the proteins separated by high resolution two-dimensional gel electrophoresis.

Amino Acid Sequence↗
PubMed1991

Detection of beta-glucanase activity on various beta-1,3 and beta-1,4-glucans after native and denaturing polyacrylamide gel electrophoresis.

beta-Glucanases were detected after polyacrylamide gel electrophoresis under native and denaturing conditions using various beta-1,3- and beta-1,4-glucans, including mixed glucans (laminarin, pachyman, carboxymethyl cellulose, lichenan and barley beta-glucan). After electrophoresis and incubation of gels, substrates incorporated into polyacrylamide gels were stained with specific fluorochromes, Sirofluor for beta-1,3 linkages and Calcofluor White M2R for beta-1,4 linkages. Under UV illumination, lysis zones appeared as dark bands against a fluorescent background. Enzymes of bacterial, fungal and plant sources could be revealed sequentially in gles containing mixed beta-(1,3)(1,4)-glucans by staining first with sirofluor followed by staining with Calcofluor White M2R. Active profiles were more diverse when substrates were stained with sirofluor. The use of purified sirofluor at pH 11.5 compared with Aniline Blue at pH 8.6 allowed better detection of beta-1,3-glucanase activities. In gels containing laminarin stained with sirofluor, bands exhibiting a more intense fluorescence than the background fluorescence were observed in addition to dark nonfluorescent bands. It is postulated that these two types of beta-1,3-glucanase activities differ by their enzymatic action (partial versus extensive hydrolysis). Analysis of fungal extracts using denaturing gels embedded with various beta-glucans displayed lysis bands migrating between 32 and 35 kDa.

Aniline Compounds↗
PubMed1991

Observation of orientation and relaxation of protein-sodium dodecyl sulfate complexes during pulsed intermittent field polyacrylamide gel electrophoresis.

Polyacrylamide gel electrophoresis (PAGE) of proteins denatured with SDS (sodium dodecyl sulfate) has been used successfully to separate proteins according to their molecular mass. In spite of the extensive use of this technique, the motion of the protein-SDS complex in a polyacrylamide gel is still not understood. Here we report on the observation of the orientation (in the field direction) and relaxation of protein-SDS complexes during pulsed intermittent field PAGE experiments. The results give an indication of the stiffness of the molecules and may be useful for the development of a technique to improve the separation of large proteins using pulsed electric fields.

Chemical Phenomena↗
PubMed1991

A novel method for specific visualisation of serum albumin in polyacrylamide gels by iodine staining.

Bovine serum, bovine serum albumins (delipidated or globulin free or Fraction V), rabbit serum, rabbit serum albumin, Atlantic salmon serum, purified Atlantic salmon serum albumin, human plasma alpha 2-macroglobulin, hemocyanin, trypsin inhibitor, bovine transferrin and bovine lactoferrin were examined by a novel method for specific visualisation of albumins. In native polyacrylamide gel electrophoresis the albumins were visualised by iodine staining as a transparent spot against a brown background whilst the other proteins could not be visualised. It is suggested that the brown background was due to penetration of the gel by iodine while the chemical binding of iodine by albumin produced a decolourisation reaction. This novel method provides a fast and simple approach to identifying serum albumin in polyacrylamide gels.

Animals↗
PubMed1991

A technique for detecting antifungal activity of proteins separated by polyacrylamide gel electrophoresis.

A technique was developed for the detection of antifungal activity of proteins after discontinuous polyacrylamide gel electrophoresis under native conditions. The antifungal activity is detected as growth inhibition zones in a homogeneous fungal lawn, grown in an agar layer spread on top of the polyacrylamide gel. The position of proteins with antifungal activity can be determined on a diffusion blot prepared from the same gel. The technique is illustrated for three antifungal plant proteins, i.e. alpha-purothionin, Urtica dioica agglutinin, and tobacco chitinase.

Antifungal Agents↗
PubMed1992

A rapid and efficient method for the screening of acid phosphatase 1 in young tomato seedlings, and for the identification of root-knot nematode species using miniaturized polyacrylamide gel electrophoresis.

A relatively rapid and highly sensitive miniaturized polyacrylamide gel electrophoresis technique is described for the analysis of certain isozymes from single cotyledons of tomato seedlings and from single females of the root-know nematode (Meloidogyne spp.). Homogenates from single tomato cotyledons (7, 14, 21, and 28 days old) were electrophoresed and stained for acid phosphatase 1 (Aps 1) activity. Cotyledons from plants of all the above age groups showed good Aps 1 activity. Nondestructive screening for tomato Aps 1 is therefore feasible, using very small samples, from as young as 7-day-old tomato seedlings. This could be of important use in expediting root-knot nematode resistance (based on the Aps 1-linked resistance gene Mi) screening for breeding programs, or F1 testing for seed production purposes. In addition, the mini-polyacrylamide gel electrophoresis technique was useful for determination of the Aps 1 allelic contribution to the total enzyme activity. The system was also used to detect malate dehydrogenase and esterase isozyme activity from single adult females of the four common root-knot nematodes, Meloidogyne arenaria, M. hapla, M. incognita, and M. javanica, with equally good results, enabling species discrimination.

Acid Phosphatase↗
PubMedDate not supplied

Photopolymerization of polyacrylamide gels with methylene blue.

Photopolymerization of polyacrylamide gels in the presence of methylene blue (100 microM) and a redox couple (1 mM sodium toluenesulfinate, a reducer, and 50 microM diphenyliodonium chloride, an oxidizer) has been investigated. The gel point, i.e. the time needed for onset of gelation upon illumination, has been found to lengthen progressively at lower temperatures and at lower light intensities. If the three catalysts are progressively diluted, the gel point does not vary for a threefold dilution, but gelation is greatly hampered below a 1:5 dilution of the three effectors. Photobleaching has been assessed as a function of liquid layer thickness (from 0.5 to 2 mm), of a progressive dye dilution (down to a fourfold dilution) and as a function of temperature. A maximum of elastic modulus is located in correspondence to a minimum of permeability (both situated at 5% cross-linker). It is found that methylene blue-activated polymerization produces polyacrylamide gels with elastic properties which are higher than in persulfate-activated gels, so far the most popular matrices for electrokinetic separations. Due to the ease of preparation, the full control of all experimental parameters, and the lack of oxidizing power of this catalyst system (as opposed to the strong oxidation power of persulfate catalysis), methylene blue catalysis is advocated as a valid alternative to other redox systems.

Acrylic Resins↗
PubMed1993

Electrotransfer of fixed phosphoproteins from pieces of dried polyacrylamide gel to small disks of nitrocellulose, nylon or polyvinylidene difluoride.

A simple method for the transfer of 32P-labeled proteins from dried polyacrylamide gels to small disks of nitrocellulose, nylon or polyvinylidene difluoride (PVDF) is described. Gel pieces containing the desired phosphoprotein are rehydrated in buffer containing sodium dodecyl sulfate (SDS) and sealed in agarose in a glass tube over a supporting gel of polyacrylamide. Protein is transferred upwards through a discontinuous density gradient of SDS-buffer and methanol to a disk of membrane sealed to the mouth of the tube with dialysis membrane. The method allows the concentration of a phosphoprotein present in several gel pieces to a single disk of immobilized membrane. Recovery of phosphoprotein was at least as good as obtained with conventional electroblotting. Application of the method to the analysis of the phosphoamino acid content of the astrocyte marker, glial fibrillary acidic protein, is described.

Amino Acids↗