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PubMed2000

Determination of PyPuPu (PyPuPy) intermolecular triple-stranded DNA by capillary electrophoresis.

The PyPuPu and PyPuPy intermolecular triple-stranded DNA (tsDNA) can be determined more easily by capillary electrophoresis (CE) than by traditional methods. The tsDNA and its component compounds can be well separated by using a sieving matrix of 1.0% hydroxypropylmethylcellulose (HPMC) containing 2.5 mM magnesium ions. Such factors as buffer pH, the concentration of triplex-forming oligonucleotide (TFO), temperature, and the concentration of magnesium cation in the formation and stabilization of triple-stranded helices have been studied with capillary electrophoresis. The triplex cannot be formed when the buffer pH is lower than 4.0. When the concentration of TFO is four times higher than that of dsDNA, all of the dsDNA molecules can be associated. The limit of capillary electrophoresis detection with good reproducibility is 0.5-1 nM (S/N = 3). The CE analysis of short tsDNA takes only 40 min, whereas gel electrophoresis needs at least 5 h.

Antineoplastic Agents↗
PubMed1995

Purification of progelatinases A and B by continuous-elution electrophoresis.

Progelatinases A and B were purified from HT1080-conditioned culture medium using a continuous-elution electrophoresis. Initially cell culture medium was ammonium sulfate precipitated. The concentrated proteins were affinity chromatographed on gelatin-Sepharose column. The bound gelatinases were eluted with electrophoresis sample buffer and subjected to continuous-elution electrophoresis. In a single run, under the standardized working conditions, the obtained fractions contained four purified enzymes--progelatinase A (M(r) 72,000), its activated forms (M(r) 62,000 and M(r) 59,000), and progelatinase B (M(r) 92,000). Moreover, the continuous-elution electrophoresis allowed the enzymes separation from their respective inhibitors--TIMP-1 (M(r) 28,500) and TIMP-2 (M(r) 21,000). The purified progelatinases A and B demonstrated high specific activities (150-200 U/micrograms).

Ammonium Sulfate↗
PubMed1990

Cadmium speciation studies in the intestine of Lumbricus terrestris by electrophoresis of metal proteins complexes.

Cadmium speciation of the intestinal compartment of the earthworm species, Lumbricus terrestris, has been investigated using polyacrylamide gel electrophoresis under non-denaturing conditions. Worms exposed to Cd(NO3)2 supplemented soils have been studied and compared to control samples. Prior to electrophoresis, the worm intestines were removed and dissected. Proteins in the crude intestinal extracts were separated using polyacrylamide gel electrophoresis. The cadmium distribution in the proteins has also been described. In a second set of experiments, cadmium bound to proteins was first isotopically exchanged with labelled cadmium (109Cd) and then cadmium speciation was performed using gel electrophoresis. Autoradiography of this gel shows an intense band in the contaminated sample whereas this band was absent in the control sample. These results show that one type of major protein has a strong affinity for cadmium in the worm intestinal extract. This type of protein had a migration close of that of rabbit liver metallothionein used for comparison.

Animals↗
PubMedDate not supplied

Characterization of latex allergenic components by capillary zone electrophoresis and N-terminal sequence analysis.

In a previous study, protein components purified from latex gloves that elicited allergenic reactions were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and yielded apparent molecular weights of 14, 22, 30, 34, 46, and 58 kD. These allergenic components were isolated for further characterization by capillary zone electrophoresis and N-terminal amino acid sequence analysis. These components all migrated at approximately 25 and 35 min on capillary zone electrophoresis. Diode array spectral analysis detected indistinguishable characteristics between these two protein peaks. In addition, complex formation of these components with patients' immunoglobulin was demonstrated by capillary zone electrophoresis. Analysis of components separated by SDS-PAGE on a polyvinylidene difluoride membrane showed that the first 13 residues were identical to the sequence of hevein. Based on the criteria of charge-to-mass ratio and N-terminal amino acid sequence, our results suggest that these components of latex proteins are similar in the primary structure.

Allergens↗
PubMed1990

Discrimination of liver cirrhosis from chronic hepatitis by analysis of serum cholinesterase isozymes using affinity electrophoresis with concanavalin A or wheat germ agglutinin.

Isozymic alteration of serum cholinesterase (ChE) was investigated in patients with chronic liver diseases using affinity electrophoresis with concanavalin A (Con A) or wheat germ agglutinin (WGA). On Con A-containing agarose gel electrophoresis, three bands with enzyme activity (named bands I to III, from the anodic side to the cathodic) were observed in sera of normal controls. Disappearance of band II was observed in 50% (15/30) of cirrhotic patients, but only one of 20 patients with chronic hepatitis lacked band II of the serum ChE isozymes. Meanwhile, WGA-containing agarose gel electrophoresis revealed that normal controls had four ChE isozymes (named bands I to IV from the anodic side to the cathodic). These four isozymes were also observed in patients with chronic hepatitis. However approximately 67% (20/30) of cirrhotic patients lacked band II of ChE isozymes. When these two affinity electrophoreses were used in combination, 22 (73%) of 30 cirrhotic patients had isozymic alteration of their serum ChE on either Con A-containing or WGA-containing agarose gel electrophoresis, or both. Thus, affinity electrophoreses with Con A and WGA seemed to be useful methods in differentiating liver cirrhosis from chronic hepatitis.

Cholinesterases↗
PubMed1995

Lectin affinity electrophoresis.

Lectin affinity electrophoresis is a powerful technique to investigate the interaction between a lectin and its ligand. Affinity electrophoresis results from the reduced mobility of a charged species owing to its interaction with an immobile species. In this protocol, a two-dimensional lectin affinity electrophoresis experiment is described that affords separation of oligosaccharides. The first-dimension is composed of a weak, polyacrylamide, capillary tube gel containing a lectin. The example described involves a mixture of fluorescently labeled disaccharides. The mobility of only the lectin-binding disaccharide is reduced affording a separation in the first-dimension. The tube gel is then extruded and placed onto the second-dimension gradient polyacrylamide gel and subjected to electrophoresis. Mobility in the second-dimension is dependent on molecular size and visualization si by fluorescence under transillumination. This method is also applicable, with appropriate modifications, for the separation and analysis of glycopeptides and glycoproteins.

Carbohydrate Sequence↗
PubMed2004

Estimation of binding constants between ristocetin and teicoplanin and peptides using on-column ligand derivatization coupled to affinity capillary electrophoresis.

This work utilizes on-column ligand synthesis and affinity capillary electrophoresis (ACE) to determine binding constants ( K(b)) of 9-flourenylmethyloxy carbonyl (Fmoc)-amino acid derivatives to the glycopeptide antibiotics ristocetin (Rist) and teicoplanin (Teic). In this technique, two separate plugs of sample are injected on to the capillary column and electrophoresed. The initial sample plug contains a D-Ala- D-Ala terminus peptide and either one or two non-interacting standard(s). The second plug contains a Fmoc-amino acid- N-hydroxysuccinimide (NHS) ester. The electrophoresis is then carried out with an increasing concentration of Rist or Teic in the running buffer. Upon electrophoresis the initial D-Ala- D-Ala peptide reacts with the Fmoc-amino acid yielding a new Fmoc-amino acid- D-Ala- D-Ala peptide derivative. Continued electrophoresis results in the binding of Rist or Teic to the Fmoc-amino acid- D-Ala- D-Ala peptide derivatives. Analysis of the change in the relative migration time ratio ( RMTR) or electrophoretic mobility (mu) of the Fmoc-amino acid- D-Ala- D-Ala peptide derivatives relative to the non-interacting standards, as a function of the concentration of Rist and Teic, yields a value for K(b). These findings demonstrate the advantage of coupling on-column ligand synthesis to ACE for estimating binding parameters between antibiotics and ligands.

Amino Acids↗
PubMed2005

Probing affinity via capillary electrophoresis: advances in 2003-2004.

This review addresses recent advances in capillary electrophoresis of biological-based molecular interaction from a broader perspective, based on applications reported during the period 2003-2004. These capillary electrophoresis-based studies of molecular interactions include affinity capillary electrophoresis, electrokinetic chromatography, and free zone electrophoresis. The review is written as a general synopsis of applications and does not cover the theory or protocol involved in the implementation of the analyses.

Carbohydrates↗
PubMed1998

Analysis of tear proteins by one- and two-dimensional thin-layer iosoelectric focusing, sodium dodecyl sulfate electrophoresis and lectin blotting. Detection of a new component: cystatin C.

BACKGROUND: Isoelectric focusing (IEF) of tear proteins has not yet been carried out in a satisfactory way. Two-dimensional (2D) electrophoresis, especially in the combination of IEF with SDS, is able to differentiate between proteins in detail. The purpose of this study was therefore to analyze tear proteins by 1D IEF alone and in combination with a 2D pattern, and by IEF followed by lectin staining. METHODS: Ampholines, covering a broad range from pH 3 to pH 10, were applied. After IEF, semi-dry blotting and incubation with a group II lectin and two group V lectins was performed. RESULTS: Tear proteins could be separated into 31 single bands. Tear-specific pre-albumin (TSPA), lactoferrin, sIgA, IgG and lysozyme were found to be main components. Isoelectric points (IEPs, pls) of all proteins separated were determined by comparison with IEF standards. 2D patterns of IEF and SDS electrophoresis were obtained for the main subunit components of lactoferrin, sIgA, TSPA, and lysozyme. An additional new component of considerable concentration was focused at pI 8.6 with a subunit MW of 14 kDa. With s-WGA a component at an IEP of 5.2 was visualized, representing transferrin. With SNA, lactoferrin stained as a sharp main band at pI 5.1 with three additional weaker bands at IEPs from 4.8 to 4.9. At IEPs between 4.4 and 6.1, multiple components of sIgA were stained with MAA. The sugar specificity of transferrin at pI 5.2 was beta-GlcNAc. Lactoferrin showed glycation with NANA-alpha-2-6-Gal or NANA-alpha-2-6-GalNAc, whereas the sugar specificity of sIgA was NANA-alpha-2-3-Gal. CONCLUSIONS: The investigative strategy applied here, including IEF alone, in combination with SDS-electrophoresis, and SDS-electrophoresis followed by lectin staining proved to be a reproducible method for tear protein analysis of hitherto unexperienced capacity. Lectin-stained bands of native tear proteins are not uniformly glycated by one sugar residue, but show various sugar specificities. IgA as a whole molecule is specifically glycated with NANA-alpha-2-3-Gal.

Adolescent↗
PubMed2003

Fractionation of liver proteins by preparative electrophoresis.

Proteomics offers unique possibilities to investigate changes in the levels and modifications of proteins involved in the pathomechanisms of diseases and toxic events. However, search for potential drug targets and disease or toxicity markers is limited by the fact that mainly the high-abundance, hydrophilic proteins are visualized in two-dimensional gels. Here we studied the enrichment of rat liver cytosolic proteins by preparative electrophoresis. Preparative electrophoresis was performed with the PrepCell apparatus in the presence of 0.1% lithium dodecyl sulfate. Lithium dodecyl sulfate was exchanged against agents compatible with isoelectric focusing prior to the two-dimensional gel electrophoresis. Proteins were identified from two-dimensional gels by matrix-assisted laser desorption ionization time-of-flight mass specrometry. Low- and middle-size proteins and low-abundance proteins, which had not been found before, were enriched by preparative electrophoresis. The present study represents a contribution of proteomics in the quantification of differences in the levels of low-abundance liver proteins in toxicity studies.

Animals↗
PubMed1984

Use of cationic detergents for polyacrylamide gel electrophoresis in multiphasic buffer systems.

An improved system for polyacrylamide gel electrophoresis in the presence of cationic detergents, cetyltrimethylammonium bromide and cetylpyridinium chloride, respectively, is described. An acidic discontinuous buffer system generated according to the theory of multiphasic zone electrophoresis developed by T. M. Jovin (1973, Biochemistry 12, 871-904) was used. It was optimized with respect to the operational conditions and to the desirable range of relative mobility values for the proteins that have molecular weights from 16,500 to 90,300. Also presented is a procedure for the elimination of interference from cationic detergents frequently encountered during staining of gels. The electrophoretic system was suitable for fractionating a wide variety of proteins. The technique can also be used to provide an alternative estimate of molecular weight. To fully account for accurate estimations, the Ferguson relationship between mobility and gel concentration and the relation of molecular weight to mobility at a single gel concentration were both considered. Examples reported in this paper include the separation and/or molecular weight determination of several common proteins, histones, and microfibrillar and myofibrillar proteins. The results suggest that electrophoresis in the presence of cationic detergents offers the same degree of reliability in analysis of most proteins as is provided by the anionic detergent sodium dodecyl sulfate electrophoresis.

Buffers↗
PubMed1987

Electrophoresis of honey: characterization of trace proteins from a complex biological matrix by silver staining.

The proteins of unconcentrated honey have been detected with the methylamine-incorporating silver stain (T. Marshall, 1984, Anal. Biochem. 136, 340-346) following sodium dodecyl sulfate-polyacrylamide gel electrophoresis or high-resolution two-dimensional electrophoresis. The former consistently reveals at least 19 protein bands in a variety of Australian honeys. Two-dimensional electrophoresis gave patterns of poor resolution but proved useful for further characterization of the major protein constituents. The protein patterns were unaffected by centrifugation of the samples prior to preparation for electrophoresis.

Electrophoresis, Polyacrylamide Gel↗
PubMed1988

Comparison of diazo-coupling, formazan, and silver staining techniques for visualizing alkaline phosphatase isoenzymes after electrophoresis in homogeneous-pore and gradient-pore polyacrylamide gels.

Three techniques for visualization of alkaline phosphatase after polyacrylamide-gel electrophoresis are compared. These are diazo-dye simultaneous coupling with the substrate sodium naphthyl phosphate and 5-chloro-2-toluene diazonium chloride; formazan precipitation with the substrate 5-bromo-4-chloro-3-indolyl phosphate and 3-[4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide; and silver staining with the substrate sodium glycerophosphate. Each staining technique was tested with gradient-pore and homogeneous-pore acrylamide-gel electrophoresis. The main factors assessed are sensitivity; separation of the human serum alkaline phosphatase isoenzymes of the liver, bone, and intestinal types; and differences in substrate affinity, as well as the complexity of each technique. Using the three techniques only minor differences in substrate affinity are evident. There is some nonspecific staining with the diazo-coupling technique but not with the formazan and silver staining techniques. The differences, in the mobility of the liver, bone, and intestinal isoenzymes achieved by homogeneous-pore gel electrophoresis are sufficient to allow them to be clearly distinguished. However, only very small differences in mobility are found with gradient-pore gel electrophoresis, but the sharper bands in this medium allow much smaller amounts of activity to be detected. As little as 160 microU of enzyme can be visualized by the diazo technique. Silver staining gives an approximately fourfold increase in sensitivity over the formazan technique, which in turn gives a fourfold increase over the diazo technique. An important aspect of the silver staining technique is the potential of increasing sensitivity much further by improvements in the photographic physical development stage.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed1988

Dodecyl maltoside-sodium dodecyl sulfate two-dimensional polyacrylamide gel electrophoresis of chloroplast thylakoid membrane proteins.

A two-dimensional electrophoretic system has been developed for the separation of chloroplast thylakoid membrane proteins. This system incorporates nondenaturing polyacrylamide gel electrophoresis in the presence of the nonionic detergent dodecyl-beta-D-maltoside in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. Thylakoid membranes isolated from Spinacia oleracea were solubilized in 1.0% dodecyl-beta-D-maltoside and separated in 4-7% linear acrylamide gradient tube gels which contained 0.05% dodecyl-beta-D-maltoside. After electrophoresis, the tube gels were equilibrated with a sodium dodecyl sulfate-containing equilibration buffer and applied to a 12.5-20% acrylamide linear gradient gel. The Lammelli buffer system was used in both dimensions. The two-dimensional gels were analyzed by staining sequentially with 3,3',5,5'-tetramethylbenzidine-H2O2, Coomassie blue, and silver staining. A number of protein components were identified on "Western blots" of these two-dimensional gels by immunological localization. Membrane protein complexes such as the light-harvesting chlorophyll a/b protein complex, photosystem I, photosystem II, the cytochrome b6/f complex and ribulose bisphosphate carboxylase appear to migrate as essentially intact complexes in the first dimension and appear as vertical series of resolved subunits in the second dimension. This technique complements isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis in providing additional information concerning the subunit composition of membrane protein complexes and may prove to be of general utility for studying the protein composition of other membrane systems.

Chloroplasts↗
PubMed1990

Detection of chitin deacetylase activity after polyacrylamide gel electrophoresis.

Mucor racemosus and Rhizopus nigricans were used as sources of chitin deacetylases. Crude protein extracts were subjected to polyacrylamide gel electrophoresis at pH 8.9 (Davis system) or 4.3 (Reisfeld system) under native conditions. After electrophoresis, an overlay gel containing 0.1% (w/v) glycol chitin as substrate was incubated in contact with the separation gel. Chitin deacetylase activity was revealed by uv illumination with a transilluminator after staining for 5 min in 0.01% (w/v) Calcofluor white M2R. Chitosan (deacetylated chitin) generated by chitin deacetylases appeared more fluorescent than the intact chitin embedded in the overlay gel. Chitosan in a separate overlay gel was also subjected to a nitrous acid treatment which specifically depolymerizes chitosan while leaving chitin intact. Hydrolysis of chitosan by nitrous acid followed by Calcofluor staining yielded dark (nonfluorescent) bands (chitin deacetylase activities) in the fluorescent chitin-containing gel. Both assays revealed the presence of several chitin deacetylases from Zygomycetes. The same assays were performed after denaturing electrophoresis in 12% (w/v) polyacrylamide gels containing 0.1% (w/v) glycol chitin. Enzymes were renatured in buffered 1% (v/v) purified Triton X-100. Chitin deacetylases with estimated molecular weights between 26,000 and 64,000 were detected after Calcofluor staining. The assays were also performed in two-dimensional gel electrophoretic systems. Chitin deacetylases can be rapidly revealed by using the assay involving the nitrous acid treatment. However, both assays (with and without nitrous acid treatment) should be run to conclusively demonstrate chitin deacetylase activity after polyacrylamide gel electrophoresis.

Amidohydrolases↗
PubMed1990

Modification of a discontinuous and highly porous sodium dodecyl sulfate-polyacrylamide gel system for minigel electrophoresis.

A highly porous and efficient discontinuous sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis system was recently described by J. P. Doucet and J. M. Trifaró [1988) Anal. Biochem. 168, 265-271). The system was developed to separate with high and broad resolution the components from large volume samples after an overnight electrophoresis. This system was found to have many advantages. However, when used directly as a minigel system, this method cannot sustain the high voltage inherent to minigel electrophoresis and produces artefacts, namely a double front and a loss of resolution in the low molecular weight range. These problems were eliminated using the buffer system of M. A. Porzio and A.M. Pearson [1977) Biochem. Biophys. Acta 490, 27-34) in the separating gel and in the electrode chambers. The resulting modified discontinuous minigel system has the same advantages as the technique described for large slab gel electrophoresis, including the effective and rapid transfer of high molecular weight proteins to nitrocellulose membranes, as well as the advantages of the minigel format.

Animals↗
PubMed1990

Staining properties of bovine low molecular weight hydrophobic surfactant proteins after polyacrylamide gel electrophoresis.

Reports describing polyacrylamide gel electrophoresis patterns of bovine hydrophobic surfactant proteins are not consistent. In this study, we found unusual staining characteristics of these proteins that may explain some of these inconsistencies. Low molecular weight surfactant proteins extracted from bronchoalveolar lavage with organic solvent are partially delipidated with Sephadex LH-20 chromatography using chloroform and methanol. Fractions from the first protein peak are dried under nitrogen then subjected to SDS electrophoresis on 20% polyacrylamide gels. Under nonreducing conditions, silver staining identifies 5- and 26-kDa bands, and Coomassie blue identifies 6-, 12-, and 26-kDa bands. When gels are stained with Coomassie blue then silver, the 5- and 26-kDa bands stain with silver and 6- and 12-kDa bands remain stained with Coomassie blue. If gels are first stained with silver then Coomassie blue, similar results occur. We modified the silver staining protocol by treating gels with dithiothreitol or 2-mercaptoethanol after electrophoresis. With this modification, 5-, 6-, 12-, 26-, and also 17-kDa bands are identifiable. Using the modified protocol and restaining gels previously stained with silver, 6-, 12-, and 17-kDa bands that were not identified previously all became visible. In further experiments, protein bands of 6-, 12-, and 26-kDa that were identified by Coomassie blue were electroeluted under nonreducing conditions. After electrophoresis of the eluted 26-kDa protein, bands of 17-, and 26-kDa under nonreducing, and 8-kDa only under reducing conditions, were apparent by using the modified silver protocol.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1980

Identification of ectoproteins of human platelets. Combination of radioactive labelling and two-dimensional electrophoresis.

Two-dimensional gel electrophoresis combining isoelectroc focussing of reduced or non-reduced proteins in the first dimension with electrophoresis in sodium dodecyl sulfate polyacrylamide gels in the second dimension enabled us to identify 25 ectoproteins in the non-reduced state and 32 in the reduced state. Gel electrophoresis in sodium dodecyl sulfate of non-reduced proteins in the first dimension followed by reduction and gel electrophoresis in sodium dodecyl sulfate in the second dimension was helpful in the identification of the major ectoproteins and indicated that at least seven additional components might be present in the region between 170 and 85 kdaltons. All major ectoproteins could readily be identified. Glycoprotein V showed only a small increase in apparent molecular weight on reduction. It was one of the most basic proteins with a pI value of 6.9. The majority of the ectoproteins were located at an isoelectric point of 5.7 with glycoproteins Ib, IIc, VI and VII as the most acidic components. A good agreement was observed between the thaee labelling techniques which indicates that almost all ectoproteins are glycoproteins containing sialic acid.

Autoradiography↗