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PubMed1993

Differentiation of proteins in polyacrylamide gels by a modification of silver staining for the PhastSystem and a laser densitometer.

Nonspecific background staining of the gel matrix is the limiting factor in the differentiation of proteins using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is caused by nonspecific binding of silver ions in the gel matrix, either to nonprotein compounds or through the chemistry of the polyacrylamide gel itself. The resulting stain of the gel produces a stain baseline, making differentiation of protein bands with a laser densitometer difficult. We have therefore developed a modified silver staining method for the PhastSystem to reduce such nonspecific background staining. Apart from slowing the development program by lowering the temperature to 15 degrees C and shortening the incubation time to 4 min, the essential step in the modification is the combined use of an EDTA and Tris-acetate buffer solution which stops the reduction of silver ions. The reduced background staining leads to an improved detection of protein bands and virtually identical zero lines for the laser densitograms and the stain baselines. The total staining time is 91 min. All steps in the program are fully automated and continuous, employing the PhastSystem staining unit.

Densitometry↗

PubMedDate not supplied

Native polyacrylamide gel electrophoresis of membrane proteins: glutaminase detection after in situ specific activity staining.

A new procedure for the analysis and detection of phosphate-activated glutaminase (EC 3.5.1.2) by native electrophoresis has been developed. The method is based on the in situ detection of glutaminase activity in two different systems of native polyacrylamide gradient gels, containing 3-(3-cholamidopropyl)-dimethyl-ammonio-1-propane sulfonate (CHAPS) or Triton X-100 as nondenaturant detergents. Crude Triton X-100 extracts of mitochondria were resolved by electrophoresis. The enzyme was specifically revealed by incubation of the gel with glutamine and coupling the oxidation of the glutamate formed to the reduction of a tetrazolium dye, in the presence of glutamate dehydrogenase trapped in a 1% agar solid overlay. Both Ehrlich ascitic cell and mouse kidney glutaminases were resolved by native electrophoresis and specifically detected with the activity staining. Moreover, the redox-cycling staining was tested in solution, showing linearity with the amount of glutamate or glutaminase activity present. The method described could be a useful tool for native polyacrylamide gel electrophoresis of membrane proteins.

Animals↗

PubMed1994

Detection of exo-beta-1,3-glucanase activity in polyacrylamide gels after electrophoresis under denaturing or nondenaturing conditions.

A method for the visualization of exo-beta-1,3-glucanase activity in polyacrylamide gels is presented. The procedure consists of the enzyme reaction in the gel with the substrate alpha-naphthylglucopyranoside, and a subsequent staining of the obtained alpha-naphthol with dyes Fast Red B, or Fast Blue BB, respectively. A mixture of exoglucanases produced by the fungus Polyporus squamosus was used for the optimization of the method. The procedure is applicable for the standard Laemmli discontinuous electrophoresis system, even in the presence of sodium dodecyl sulfate, as well as for electrophoresis in linear gradients of the polyacrylamide concentration. The staining method was used for the analysis of exoglucanases secreted by several yeast genera. All yeasts tested produced two types of exoglucanases, a high molecular mass species heterogeneous in size, and one or two smaller homogeneous enzymes.

Densitometry↗

PubMed1994

Horizontal polyacrylamide gel electrophoresis for the separation of DNA fragments.

A discontinuous borate/formate buffer system is presented for horizontal polyacrylamide gel electrophoresis of DNA fragments. The resolution potential of the system could be altered by changing the total monomer concentration (5-9%T), the concentration of the crosslinker piperazine diacrylamide (2-5%CPDA), as well as the concentration of formate in the gel (40-120 mM), the leading ion of the buffer system. The separation of DNA fragments would be improved by increasing the migration distance from 22 to 28 cm. This discontinuous polyacrylamide gel electrophoresis system proved highly reproducible.

Buffers↗

PubMed1995

Effective blotting of ultrathin polyacrylamide gels anchored to a solid matrix.

Ultrathin polyacrylamide gels bound on glass plates or plastic sheets cannot be removed from their support without destruction. Therefore electrophoretic transfer methods are not applicable. We have developed a fast diffusion blotting procedure which is very simple and does not need any equipment like blotting chamber or power supply. Furthermore, no special buffer solutions are required. The method is universally applicable to ultrathin sodium dodecyl sulfate, native as well as isoelectric focusing polyacrylamide gels.

Acrylic Resins↗

PubMed1995

The influence of proteins on silver staining of nucleic acids following polyacrylamide gel electrophoresis.

The influence, on silver staining, of proteins and restriction enzymes in polymerase chain reaction (PCR) products was studied in 12-20% polyacrylamide gels. For small DNA fragments (74, 41 and 33 bp) best results were achieved using 20% polyacrylamide gels. In 12% gels, restriction enzymes and also proteins used for PCR (bovine serum albumin, Taq-DNA polymerase) interfere with silver staining of nucleic acids.

DNA↗

PubMed1995

Single-step electrotransfer of reverse-stained proteins from sodium dodecyl sulfate-polyacrylamide gel onto reversed-phase minicartridge and subsequent desalting and elution with a conventional high-performance liquid chromatography gradient system for analysis.

Isolation of proteins from polyacrylamide electrophoresis gels by a novel combination of techniques is described. A given protein band from a reverse stained (imidazol-sodium dodecyl sulfate--zinc salts) gel can be directly electrotransferred onto a reversed-phase chromatographic support, packed in a self-made minicartridge (2 mm in thickness, 8 mm in internal diameter, made of inert polymeric materials). The minicartridge is then connected to a high-performance liquid chromatography system and the electrotransferred protein eluted by applying an acetonitrile gradient. Proteins elute in a small volume ( < 700 microL) of high-purity volatile solvents (water, trifluoroacetic acid, acetonitrile) and are free of contaminants (gel contaminants, salts, etc). Electrotransferred proteins were efficiently retained, e.g., up to 90% for radioiodinated alpha-lactalbumin, by the octadecyl matrix, and their recovery on elution from the minicartridge was in the range typical for this type of chromatographic support, e.g., 73% for alpha-lactalbumin. The technique was successfully applied to a variety of proteins in the molecular mass range 6-68 kDa, and with amounts between 50 and 2000 pmol. The good mechanical and chemical stability of the developed minicartridges, during electrotransfer and chromatography, allowed their repeated use. This new technique permitted a single-step separation of two proteins unresolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis due to their different elution from the reversed-phase support. The isolated proteins were amenable to analysis by N-terminal sequencing, enzymic digestion and mass spectrometry of their proteolytic fragments. Chromatographic elution of proteins from the reversed-phase mini-cartridge was apparently independent of the specific loading mode employed, i.e., loading by conventional loop injection or by electrotransfer.

Animals↗

PubMed1995

Direct detection of beta-1,3-glucanase in plant extracts by polyacrylamide gel electrophoresis.

By using carboxymethyl (CM)-curdlan, a polysaccharide linked with the dye Remazol Brilliant Blue (RBB) as a substrate in polyacrylamide gels, the beta-1,3-glucanase in plant extracts can be detected directly by native polyacrylamide gel electrophoresis. In contrast to the usually used procedures for the detection of glucanases, e.g., colorimetric assay, overlay technique, enzyme activity staining using laminarin as a substrate, this method is rapid and allows both the determination of the activity and the location of the relative position of the multiple forms of beta-1,3-glucanases.

Animals↗

PubMed1995

High-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunochemical identification of the 2X and embryonic myosin heavy chains in complex mixtures of isomyosins.

In mammals myosin heavy chains (MHC) are polypeptides with a molecular mass of about 200 kDa whose isoforms can be identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunochemistry. Electrophoretic analysis is the only method for quantitating MHC profiles in single myofibers and/or cryostat sections of biopsied muscle. We present a method for SDS-PAGE of adult rat skeletal muscle which resolves MHC into four bands: 1, 2B, 2X, and 2A from the faster to the slower migrating band. Furthermore, embryonic MHC can be also resolved in a complex mixture of isomyosins, e.g. developing or regenerating muscles. The method does not involve preparation of gradient gels or electrophoresis at low temperature. Improved reproducibility is obtained by: (i) modification of the sample buffer; (ii) use of 7% polyacrylamide in the separating gel; (iii) control of pH of running buffer by recirculation or change of the buffer during the run; and (iv) a 24 h run. The procedure is compatible with Coomassie Brilliant Blue, silver and immunoblot staining. Resolution is sufficient to permit transblotting of separated MHC after SDS-PAGE. The different isoforms are easily identified with monoclonal antibodies. The technique provides an improved method to separate MHC and quantitate MHC2X and MHCemb in complex mixtures of MHC from a few cryostat sections of normal and diseased muscle.

Animals↗

PubMed1995

Chemical stability of polyacrylamide-coating on fused silica capillary.

The chemical stability of polyacrylamide coatings on a fused silica capillary for capillary electrophoresis was investigated with respect to temperature of capillary and pH as well as salt concentration of running buffer solution by evaluating their effects on electroosmotic mobility. The capillary covalently bonded to linear polyacrylamide through Si-C linkage was stable in buffer solutions of pH 2.3-8.0 at 30 degrees C for 30 days, whereas the capillary bonded through Si-O-Si-C linkage was damaged at pH above 4.6. The electroosmotic mobility observed in both capillaries increased with increasing temperature, though the degree of increase was smaller in the Si-C linked capillary than in the Si-O-Si-C linked capillary. The increase in the buffer concentration resulted in increased electroosmotic mobility in the Si-O-Si-C linked capillary, but no effect was observed for the Si-C linked capillary.

Acrylic Resins↗

PubMed1996

Detection and quantitation of specific mRNAs by ribonuclease protection assay using denaturing horizontal polyacrylamide gel electrophoresis: a radioactive and nonradioactive approach.

A radioactive (32P) and nonradioactive (digoxigenin) ribonuclease protection assay (RPA) has been developed to detect mRNAs of housekeeping proteins and growth factors. A modification of polyacrylamide gel electrophoresis (PAGE) to simplify RPA is described. Both Cleangels (Pharmacia) and laboratory-cast polyacrylamide gels, in a denaturing, horizontal electrophoresis system, were used. The amount of toxic chemicals and waste was reduced, in comparison with sequencing gels normally used for RPA. The protected RNA fragments were shown to be well-separated, with sufficient sensitivity in this modified, quick gel system.

Animals↗

PubMed1997

Analysis of toxic drugs by capillary electrophoresis using polyacrylamide-coated columns.

Toxic drugs, including barbiturates and benzodiazepines, were analyzed using polyacrylamide-coated columns in capillary electrophoresis (CE). The separations were carried out in absence of electroosmotic flow. Seven kinds of barbiturates were successfully separated with the coated columns in free solution without further additives. Benzodiazepines, the electrically neutral solutes were introduced onto the coated column, and separated in presence of sodium dodecyl sulfate above its critical micelle concentration in the running buffer. This CE method offered fast and efficient separation of more hydrophobic solutes, such as benzodiazepines. The separation of seven barbiturates was studied in linear (noncross-linked) polyacrylamide solutions and in acrylamide/N-isopropylacrylamide (AA/IPAA) copolymer solutions to explore the effect of isopropyl groups in the AA/IPAA copolymer chain.

Acrylic Resins↗

PubMed1997

Detection and semiquantitation of albumin forms in fresh human plasma separated on gradient polyacrylamide gel by means of electroblotting on agarose gel matrix.

Albumin in vitro contains several molecular forms, while in vivo it exists mainly as a monomer with a small fraction of a dimer. The aim of the present work was to detect and estimate albumin forms in fresh blood samples. The available analytical methods at present are inadequate for this purpose. An improved immunoblotting method was used where plasma was subjected to electrophoretic separation on 4-25% gradient polyacrylamide gels followed by immunoblotting on agarose gel containing anti-human albumin. The interference from the huge amount of the monomer in plasma was overcome by cutting the monomer region from the polyacrylamide gel before immunoblotting. After staining of the agarose gel, it revealed the presence of seven stained bands of albumin in addition to the monomer. These bands represent albumin aggregates and complexes of varying molecular masses (112-428 kDa). These albumin forms accounted for 0.7% of the total plasma albumin and their estimated level was 30.7 mg/dL. This study shows that the native albumin in blood has several molecular forms. It is concluded that albumin in healthy human subjects may form association complexes of varying molecular masses with other macromolecules in blood and these complexes are expected to be of physiological relevance.

Electrophoresis, Polyacrylamide Gel↗

PubMed1998

Ultrathin-layer zone electrophoresis of alcohol dehydrogenase in partly rehydrated polyacrylamide gels: an alternative to starch gel electrophoresis.

A highly sensitive electrophoretic technique for the separation of alcohol dehydrogenase isoenzymes by zone electrophoresis in partly rehydrated polyacrylamide gels is described. Five hundred microm thin polyacrylamide gels are polymerized under standardized conditions. After polymerization the gels are washed thoroughly with distilled water to remove any unreacted monomers, catalysts or still soluble polymers. The washed gels are then impregnated with 0.5% Tween 20 and dried. Before electrophoresis the dry gels are rehydrated to a thickness of 250 microm, which makes up 50% of the original gel volume. Rehydration is carried out by use of a degassed buffer solution. This method permits the demonstration of the isoenzymes of alcohol-dehydrogenase class I and II in man and allows quantitative determination.

Acrylic Resins↗

PubMed1998

Isolation and characterization of beta-cyclodextrin sulfates by preparative gradient polyacrylamide gel electrophoresis, capillary electrophoresis and electrospray ionization - mass spectrometry.

A beta-cyclodextrin sulfate mixture has been fractionated using discontinuous gradient polyacrylamide gel electrophoresis. Semidry electrotransfer of the sample onto a positively charged nylon membrane and visualization of a portion of this membrane with Alcian blue stain showed multiple bands. The bands were cut from the remaining portion of the membrane and after washing with 8 M urea, the beta-cyclodextrin sulfate fractions were eluted with 2 M sodium chloride and dialyzed. Analysis of each fraction using high resolution analytical gradient polyacrylamide gel electrophoresis as well as capillary electrophoresis, using indirect detection, showed some of the fractions to be pure while others were mixtures. Each beta-cyclodextrin sulfate fraction was complexed with a basic synthetic peptide and analyzed by electrospray ionization mass spectrometry to define the mass of the components in each mixture and thereby to determine the purity of each sample.

Carbohydrate Sequence↗

PubMed1998

Preparative application of commercial automated gel electrophoresis apparatus to subcellular-sized particles: sequential isolations, fractions re-run, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, yield and purity.

The analytical and preparative potential of automated gel electrophoresis apparatus with intermittent fluorescence scanning of the migration path, the HPGE-1000 apparatus (LabIntelligence, Belmont, CA) was further developed in application to subcellular-sized particles. Resolution between two rat liver microsome components in agarose (MetaPhor) gel electrophoresis was found to increase with decreasing agarose concentration to 0.04%. It was less, even in an agarose solution at that low concentration, than that in laterally aggregated 4% polyacrylamide gel. The three components of the microsomal preparation were sequentially isolated from 0.6 and 0.8% agarose gel electropherograms. One fraction when re-electrophoresed was found to exhibit the original mobility and did not give rise to the other components. Yields of each component were near-quantitative after one or two electroelution steps. Based on protein content, no impurities could be detected in two of the microsome fractions; the third fraction contained 2% of nonmicrosome impurity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of all three microsome fractions were indistinguishable from one another and from that of the unfractionated microsome preparation.

Animals↗

PubMed2003

Use of high-molecular-mass polyacrylamides as matrices for microchip electrophoresis of DNA fragments.

DNA fragment analysis requires the use of polymer solutions as sieving matrices. Generally, such matrices are constituted of high-molar-weight polymers employed at a concentration higher than their entanglement threshold concentration. These polymer solutions are highly viscous and difficult to use in the narrow channels of a microchip. Ultralarge polyacrylamides synthesized via a nonconventional method, being the low-temperature plasma-induced polymerization (PIP), were used as DNA sieving matrices for microchip electrophoresis. The distinctive features of these polymers (ultralarge molecular mass and linearity) allow their use at a dilute concentration. Dilute PIP polyacrylamides revealed a constant value of resolution in a broad range of DNA fragment sizes (123 bp-1353 bp), thus proving to be effective in common genotyping applications. Moreover, the low viscosity of the dilute solutions enable it to be easier and faster in filling the channel between runs, thus enhancing the throughput of the microchip devices.

Acrylic Resins↗

PubMed2004

Polyacrylamide gel electrophoresis of fluorophore-labeled hyaluronan and chondroitin sulfate disaccharides: application to the analysis in cells and tissues.

This report describes a new formulation of polyacrylamide gel electrophoresis of fluorophore-labeled saccharides (PAGEFS) for the analysis of hyaluronan (HA) and chondroitin sulfate (CS) Delta-disaccharides. PAGEFS relies on derivatization of reducing ends of HA- and the variously sulfated CS-derived Delta-disaccharides with 2-aminoacridone (AMAC), followed by electrophoresis under optimized buffer conditions (Tris-borate and Tris-HCl) and on polyacrylamide gels (25% T/3.75% C). The method was applied to the analysis of glycosaminoglycans (GAGs) from the human umbilical cord tissue and GAGs isolated from human aortic smooth muscle cell cultures. The obtained results were in agreement with those obtained after an analysis with high-performance liquid chromatography (HPLC). On the basis of these results, PAGEFS is a rapid and sensitive method for the analysis of the total amount of HA- and CS-derived disaccharides, as it allows analyzing 20 samples in minigels in one run and provides quantitation with relatively high sensitivity (less than 25 pmol per disaccharide). In addition, PAGEFS overcomes the lack of commercial gels described previously for the separation of AMAC-labeled disaccharides. Therefore, the method proposed here is an economic and useful tool for a fast screening of GAGs in biological samples, particularly when a high number of samples should be analyzed.

Aorta↗