bluuebunny/crossref_metadata_2025_split huggingface dataset:
Heterogeneous nature of human complement factor B: An electrophoretic approach for the analysis of its oligosaccharide chain structure and its physiological breakdown products
Authors: Garnier Gérard , Davrinche Christian , Charlionet Roland 
Published: May 2005

AbstractFactor B is a glycoprotein which plays an essential role in the alternative pathway of complement activation. It carries the proteolytic activity of the convertases, and its physiological breakdown products Ba and Bb have some effects on the cells of the immune system. Human factor B exhibits a microheterogeneity and five isoforms are present in serum. The nature and origin of the microheterogeneity was investigated by using electrophoretic techniques. Treatments of factor B with neuraminidase and glycopeptidase F show that this microheterogeneity is mainly due to differences in its sialic acid content, varying from seven to eleven residues per molecule, and resulting in different oligosaccharide structures. However, deglycosylated factor B reveals a residual, nonallotypic variation in the Bb region of the polypeptide backbone. We confirm the presence of four asparagine‐linked oligosaccharide chains of the complex type in native factor B, two of which are located in the Ba fragment, and the two others in the Bb fragment. The prevalent isoform of the native protein carries two sialic acid residues per oligosaccharide chain. Biosynthesis experiments show that the microheterogeneity of secreted factor B from HepG2 cells is acquired during the processing of its glycans. However, in vitro‐secreted factor B is more heterogeneous than the serum protein. We propose a structural model for the microheterogeneity of the native protein and its physiological fragments. We discuss as well the feasibility of electrophoretic techniques to deal with microheterogeneity analysis.
Ultrasensitive polychromatic silver staining of sodium dodecyl sulfate‐polyacrylamide gels with the Gelcode system using the PhastSystem Development Unit
Authors: Schiff Theodore A. , Freedberg Irwin M. 
Published: May 2005

AbstractThe Gelcode color‐based silver staining system, an improved formulation based on the original publication by Sammons et al. (Electrophoresis 1981, 2, 141–147) has been adapted to automated rapid staining in the PhastSystem Development Unit. The use of elevated temperatures in the fixation, washing, staining, and stabilization steps of the protocol reduces the total time of the process from 18 h to 1 h. The limit of detection, which is at least tenfold more sensitive than the silver staining protocol recommended for the PhastSystem, corresponds to 0.05–0.1 ng of protein per band. The method is applicable to both one‐ and two‐dimensional polyacrylamide gels.
Specific polypeptide differences in normal versus malignant breast tissue by two‐dimensional electrophoresis
Authors: Wirth Peter J. 
Published: May 2005

AbstractHigh resolution two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) in combination with computer‐assisted densitometry was used to analyze 800–1000 silver stained postmitochrondrial and 600–800 cytosolic polypeptides extracted from malignant and nonmalignant human breast tissues. The 2D‐PAGE patterns of polypeptides from malignant and normal tissues were similar, although both qualitative and quantitative polypeptide differences were noted. Six cytosolic poly peptides (pI/ molecular mass X 10−3), 5.20/80, 5.75/43, 6.20/40, 5.43/35, 5.46/34.5, and 5.50/34 were detected exclusively in malignant tissues. One constitutive poly peptide, p52 (7.25/52), was not detected in tumor samples. Marked quantitative differences in spot density were noted in polypeptides localized mainly in the molecular weight ranges of 22–40 kDa and pI of 5.65–7.00. An overall increase in polypeptide expression was noted in this region of 2D‐PAGE gels of malignant tissues as compared to normal. Twenty‐two acidic and 19 polypeptides separated under nonequilibrium isoelectric focusing conditions were significantly increased in tumor samples while one polypeptide was decreased. One polypeptide, p24 (6.15/24), was expressed in greatest concentrations in tumors which also expressed the greatest estrogen receptor content. Expression of p24 was markedly reduced in normal tissue and in malignant tissues expressing low levels of estrogen and progesterone receptors. No significant differences in the expression of the Yb and Ya subunits of glutathione S‐transferases (GST)‐A, ‐B and ligandin were observed between normal and malignant breast tissue. None of the Yp subunits of the placental isoform of GST were detected in either normal or malignant breast tissues.
A procedure for analysis of densitometric spectra
Authors: Galat Andrzej 
Published: May 2005

AbstractComputer‐aided quantitative analysis of densitometric spectra is presented. The densitometric spectra are decomposed into component bands using the Powell and Marquardt minimizers. Several different functions for the component bands are utilized. It is shown that the densitometric spectra can be decomposed into component bands with high accuracy only if the proper shapes of the bands are chosen. The method described was used for quantitative analysis of densitometric spectra of DNA cleaved by neocarzinostatin. The procedure is general and can be applied to analyses of autoradiographic films and to direct scans of electrophoretic gels. It is shown that densitometric spectra which show highly overlapped bands are well approximated by asymmetric Cauchy and Gauss functions. Well separated densitometric bands which have substantial asymmetry can be fitted to more sophisticated shapes formed by combinations of symmetric and asymmetric Cauchy and Gauss functions. High accuracy fitting to asymmetric densitometric curves may only be achieved using the cosine function introduced by Mignot and Rondot, J. Appl. Cryst. 1976, 9, 460–465.
Distortion of the electrophoretic titration curves of some proteins
Authors: Suttnar Jiří , Dyr Jan Evangelista 
Published: May 2005

AbstractThe electrophoretic titration curves of complex mixtures of vitamin K‐dependent human blood proteins and proteins of Bothrops asper venom were investigated. In both protein mixtures some curves exhibited marked distortions such as additional maxima and minima when Pharmalyte 3–10 carrier ampholytes were used for isoelectric focusing in agarose gels. The distortions result from an unspecific interactions‐between some carrier ampholyte constituents with particular proteins. The interacting carrier ampholyte components could be completely removed by binding to albumin and ultrafiltration through a UM‐2 Amicon membrane with resultant regular titration curves. The interacting carrier ampholyte species were only partially removed by ultrafiltration through a UM‐2 membrane without incubation with albumin.
Isotachophoretic determination of herbicides prometryne, desmetryne, terbutryne and hydroxy‐derivatives of atrazine and simazine in extracts of milk
Authors: Křivánkova Ludmila , Boček Petr , Tekel Jozef , Kovačičová Jana 
Published: May 2005

AbstractA method is described for the determination of the triazine herbicides prometryne, desmetryne, terbutryne, OH‐atrazine and OH‐simazine in purified extracts of milk using analytical capillary isotachophoresis. The reproducibility of isotachophoretic analyses was 3.5% and the detection sensitivity reached 2 ng. Recovery of triazines from fortified samples of homogenized full milk (0.05 mg/L) was about 65%.
Study of grasshopper meiosis‐associated proteins using two‐dimensional polyacrylamide gel electrophoresis
Authors: Percy Melanie J. , Fletcher Hugh L. 
Published: May 2005

AbstractPremeiotic and meiotic whole testes from grasshoppers were compared for the presence of meiosis associated proteins using one‐ and two‐dimensional sodium dodecyl sulphate‐polyacrylamide gel electrophoresis. One‐dimensional sodium dodecyl sulphate‐polyacrylamide gels detected differences between premeiotic and meiotic samples but two‐dimensional gels gave more precise results. Isoelectric focusing revealed only one meiosis‐associated protein, while nonequilibrium pH gradient electrophoresis detected five more. It is not known whether these proteins relate to the nuclear aspects of meiosis, or associated cellular changes. These proteins have been electrophoretically purified and monoclonal antibodies are being prepared.
The PhastSystem equipment used for crossed immunoelectrophoresis combined with immunoblotting of coprecipitated monoclonal antibodies as studied with platelet membrane receptor proteins
Authors: Solum Nils Olav , Aakhus Anne‐Margrethe , Pedersen Turid , Gaudernack Gustav 
Published: May 2005

AbstractThe Pharmacia PhastSystem equipment has been used for crossed immunoelectro‐phoresis combined with a technique for immunoblotting with monoclonal antibodies. This miniaturized gel system is compared to the conventional approach using platelet membrane receptor proteins as a model. Whereas in the conventional system the electrophoretic procedure takes place within 20 h, 3 h are adequate for the small gel system. Because of the short second‐dimensional electrophoresis, and only one over‐night incubation, the total electrophoretic and blotting procedure could be reduced from about 48 h to 24 h. The amount of antiserum used during the second‐dimensional electrophoresis could be reduced roughly by a factor of 5. The examples with electrophoresis and immunoblotting using platelet extracts in 1 % Triton X‐100 demonstrate that membrane receptor proteins can be studied even when present as noncovalent complexes. The immunoblotting can be used with monoclonal antibodies that do not function in Western blotting.
Polyacrylamide gel electrophoretic characterization of isoenzymes in the marine algal genus Callithamnion
Authors: Russell Stephen J. 
Published: May 2005

AbstractTechniques have been developed for the visualisation of a range of enzyme species extracted from field collected marine algal populations, to provide biochemical data in the form of zymogram patterns that closely reflect the genetic identity of these plants. These patterns provide an alternative basis for increasingly accurate recognition to complement that provided by morphology, thus helping to clarify the taxonomic problems which exist in the marine algal genus Callithamnion Lyngbye as currently conceived.
Determination of 4‐methylimidazole in caramel color by capillary isotachophoresis
Authors: Kvasnička František 
Published: May 2005

AbstractA method for the determination or 4‐methylimidazole in caramel color, based on cationic separation of the sample by capillary isotachophoresis, is described. No pretreatment of the sample is necessary and the detection limit was found to be 5 ppm.
High performance capillary electrophoresis of calmodulin
Authors: Chan Kai‐Foon J. , Chen Wilbur H. 
Published: May 2005

AbstractThe electrophoretic properties of purified calmodulin were investigated. High performance capillary electrophoresis of this Ca2+‐binding protein in free solution at pH 2.5 resulted in an elution of a single peak with a retention time of approximately 4.7 min. Addition of [ethylene‐bis(oxyethylenenitrilo)]N, N, N', N'‐tetraacetic acid (EGTA) to the protein prior to capillary electrophoresis completely abolished this electrophoretic profile. Polyacrylamide gel electrophoresis of calmodulin under denaturing and nondenaturing conditions also revealed a single polypeptide band. However, the relative electrophoretic mobilities of this protein could vary, depending on the presence or absence of Ca2+. The pI of calmodulin was estimated to be 3.7 by using isoelectric focusing techniques. Analysis of this acidic protein by high performance capillary electrophoresis at pH 8.0 revealed that it could be resolved into two major and one minor polypeptide peaks, regardless of the presence or absence of Ca2+. These findings suggest that capillary electrophoresis at near physiological pH may differentiate the microheterogeneity of calmodulin.
Electrophoretic separation of protein kinases: Altered mobility with different crosslinking agents in the presence of certain detergents
Authors: Sprott Susan , Hammond Kathryn D. , Savage Nerina 
Published: May 2005

AbstractNondenaturing polyacrylamide gel electrophoresis was used to separate protein kinases in crude extracts and subcellular fractions of murine erythroleukemic cells. The kinases were detected using an in situ phosphorylation assay. The electrophoretic patterns obtained using gel bound to GelBond and prepared with AcrylAide differed from those seen without GelBond and with N, N'‐methylenebisacrylamide as cross‐linker. In an attempt to improve the resolution of the bands in the membrane fractions, detergent‐treated preparations were electrophoresed through gels which contained either 0.1 % Triton X‐100 or 0.1 % Nonidet P‐40. The resolution of the bands in this fraction was not, however, improved with the inclusion of the nonionic detergent in the gels. When cytosol was electrophoresed through gels containing detergent, a major band of cAMP‐dependent protein kinase activity showed a marked shift in mobility. This may have been the result of a structural change, altering the shape and possibly affecting the charge on the molecule, or the enzyme may have formed aggregates with the detergent.
Reassessment of commercially available molecular weight standards for peptide sodium dodecyl sulfatepolyacrylamide gel electrophoresis using electroblotting and microsequencing
Authors: Sallantin Marc , Huet Jean‐Claude , Demarteau Claude , Pernollet Jean‐Claude 
Published: May 2005

AbstractMyoglobin CNBr peptides, constituting the commercially available molecular weight calibration kits for sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, were analyzed by microsequencing after electroblotting on polyvinylidene difluoride (Immobilon) membranes. An obious disagreement was found between peptide identification and the data provided by the manufacturers. We observed 6 peptides from Mr 2500 to 17 000 corresponding, in increasing size order, to the 3 peptides resulting from the total CNBr digestion, to 2 incompletely cleaved peptides and to the intact myoglobin. Using a corrected calibration curve, a linear relationship was established from Mr 6000 to 43 000 and a second one for shorter peptides. This method of electrophoresis and electroblotting, easily adapted for peptides, is a powerful tool for peptide identification correlated with size determination. It is especially useful for CNBr‐cleaved peptides.
Microheterogeneity of human erythrocyte pyruvate kinase: Application of immunological visualization techniques after isoelectric focusing in ultrathin gels
Authors: Megow Dirk , Jacobasch Gisela 
Published: May 2005

AbstractImmunological methods for protein visualization afford high sensitivity, good specificity and resolution. We used the indirect immunoassay with peroxidase‐ and 125I‐labelled antibodies to investigate the microheterogeneity of pyruvate kinase from human red blood cells and can show an enzyme pattern with more than 10 single bands. Different methods were tested as to their applicability for protein fixation and subsequent immunological visualization in polyacrylamide gels. Besides the Western blot, immunofixation and ethanolic fixation can be recommended as fixation techniques, especially after isoelectric focusing in ultrathin gels.
Molecular characterization of pregnancy‐associated plasma protein‐A by electrophoresis
Authors: Sinosich Michael J. 
Published: May 2005

AbstractGradient polyacrylamide gel electrophoresis, isoelectric focusing and multidimensional immunoelectrophoretic techniques have been applied in order to physicochemically characterize pregnancy‐associated plasma protein‐A (PAPP‐A). By lectin affinity immunoelectrophoresis, PAPP‐A contained sialic acid, glucose/mannose and N‐acetyl‐α‐D‐galactosamine. Immunoelectrophoretic analyses after incubation with various glycolases confirmed these findings and demonstrated that PAPP‐A contained glucuronic acid, perhaps in chondroitin sulphate moities, thus indicating that PAPP‐A may be a proteoglycan rather than a glycoprotein. Analysis by metal chelate and dye ligand affinity immunoelectrophoresis demonstrated many similarities between PAPP‐A and α2‐macroglobulin (α2M). However, unlike α2M, PAPP‐A did not form immunologically reactive complexes when incubated with proteases. Furthermore, as demonstrated by autoradiographic studies, PAPP‐A did not contain internal thiolester groups, thus indicating that PAPP‐A cannot inhibit proteases by molecular entrapment and, despite the homotetrameric molecular conformation, PAPP‐A and α2M may not have evolved from a common ancestral protein.
Horizontal two‐dimensional electrophoresis of cerebrospinal fluid proteins with immobilized pH gradients in the first dimension
Authors: Mattila Kari , Pirttilä Tuula , Frey Harry 
Published: May 2005

AbstractA horizontal two‐dimensional electrophoresis method with immobilized pH gradient isoelectric focusing supplemented with carrier ampholytes in the first dimension was applied to cerebrospinal fluid (CSF) proteins. About 300 protein spots could be detected on the silver‐stained two‐dimensional maps of CSF samples. This highresolution method is a tool worthy of consideration for the research of CSF proteins and disease‐specific changes in different neurological disorders.
Alternative methods for fixing and staining gliadins in polyacrylamide gels
Authors: Clements Robert L. 
Published: May 2005

AbstractStaining efficiencies of Coomassie Brilliant BlueG‐250 (CBB G‐250) and Coomassie Brilliant Blue R‐250 (CBB R‐250) in various media were studied in efforts to reduce or eliminate requirements for trichloroacetic acid (TCA). Stained gels were compared with gels stained with CBB R‐250 in 12% TCA and evaluated for overall stain and background. Because of qualitative effects, stain intensities of low‐ and highmobility gliadins were also evaluated. Results indicated gliadins are fixed under a wide range of conditions, permitting adjustment of conditions to provide optimum staining. CBB G‐250 and R‐250 in tap water fixed and stained most gliadins. Best results were obtained with CBB G‐250 in 2% TCA, in 2% TCA containing 5% sodium sulfate, and in 2% and 5% phosphoric acid containing 5% sodium chloride or 5% sodium sulfate. Gels stained in these media were more easily observed during staining and more easily destained than gels stained in CBB R‐250 in 12% TCA.
Column isoelectric focusing in natural pH gradients generated by biological buffers
Authors: Vaidya Narendra R. , Gothoskar Balwant P. , Banerji Ashutosh P. 
Published: May 2005

AbstractUsing 2 or 3 simple Good z witterionic buffers at a 16 or 18 mmol/L final column concentration of the mixture, natural pH gradients of 4 to 8 and 3 to 9.5, respecitively, were generated in a liquid LKB column. The pH gradients, stabilized by an anticonvective sucrose gradient, were linear, reproducible and stable in the electric field up to 5h. The pH gradients were used for isoelectric focusing of a number of impure proteins such as human hemoglobin, bovine serum albumin and chicken egg white lysozyme. The protein components could be well separated in the gradient, were easily recovered and appeared to be quite pure when analyzed by sodium dodecyl sulfategel electrophoresis. Furthermore, the pH gradient 4–8 was effectively used to isolate one of the acidic isozyme (pI 5.6) components of mouse liver alcohol dehydrogenase (EC 1.1.1.1) in an enzymatically active state, suggesting that the procedure does not denature proteins. The low cost, the ease with which the pH gradients are formed, their linearity, stability for a sufficient period to allow proteins to reach equilibrium and their subsequent recovery from buffer eluates should make the procedure interesting for electrofocusing of proteins.
Isotachophoretic zone formation of serum albumin in different free fluid electrophoresis instruments
Authors: Thormann Wolfgang , Firestone Millicent A. , Sloan Jeffrey E. , Long Terry D. , Mosher Richard A. 
Published: May 2005

AbstractThe isotachophoretic behavior of a model protein, serum albumin, was examined (i) by computer simulation, (ii) by capillary isotachophoresis in HPE 100 and Tachophor 2127, (iii) by continuous flow isotachophoresis in Elphor VaP 22 and the BIO‐STREAM Separator and (iv) by recycling isotachophoresis in an apparatus of our own design. Variations in monitored zone shapes can be explained by differences in engineering aspects and fluid stabilization principles of the instruments.
Phenotyping of erythrocyte acid phosphatase and esterase D by high field strength isoelectric focusing on cellulose acetate membrane
Authors: Kane Masateru , Yamamoto Yoshio , Yamada Mitsuko , Fukunaga Tatsushige , Tatsuno Yoahitsugu 
Published: May 2005

AbstractPhenotyping of erythrocyte acid phosphatase (EAP) and esterase D (ESD) by cellulose acetate membrane isoelectric focusing (CAM‐IEF) under a nonequilibrium condition is described. In an attempt to improve the method of CAM‐IEF, we shortened the electrode distance to provide a higher field strength at a given (low) voltage. Various carrier ampholytes for EAP typing and various chemical separators for ESD typing were also tested. Good separations were obtained after 30 min IEF for EAP typing and 25 min for ESD typing. When applied to blood stains and stored for various periods at room temperature, the stains up to 8 months old could still be phenotyped for EAP and those up to 4 weeks old for ESD. CAM‐IEF is suitable for routine forensic work of EAP and ESD phenotyping.
Pouring wide‐range immobilized pH gradient gels with a window of extremely flattened slope
Authors: Altland Klaus , Altland Alexander 
Published: May 2005

AbstractA gradient mixing device has been designed to pour polyacrylamide gels with a widerange immobilized pH gradient including a „window”︁ of extremely flattened slope. The device consists of an IBM‐compatible personal computer controlling 8 step‐motor‐driven burettes, with four producing a density gradient from glycerol and delivering acrylamides and catalysts for gel polymerization, two delivering Immobilines for a wide‐range pH gradient and the other two burettes responsible for the delivery of Immobilines for a partial pH range inside the wide range. The effect on a complex separation pattern of proteins with a wide range of pI is that resolution can be increased reproducibly to any reasonable extent at any location of the separation pattern.
Deoxyribonuclease zymography adapted to the precast PhastGel electrophoresis media
Authors: Strætkvern Knut O. , Raae Arnt J. 
Published: May 2005

AbstractA set‐up for casting fluorescent indicator agarose gels on ultrathin polyacrylamide microelectrophoresis gel media (Pharmacia PhastGel media) is described. The zymogram system allows a rapid and sensitive detection of deoxyribonuclease in various gel media, following isoelectric focusing, native and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis.
Image analysis techniques for automatic evaluation of two‐dimensional electrophoresis
Authors: Häder Donat‐P. , Kauer Gerhard 
Published: May 2005

AbstractTechniques for automatic analysis of two‐dimensional electrophoresis gels by computer‐aided image analysis are described. Original gels or photographic films are scanned using a laser scanner and the files are transferred to a microcomputer. The program package first performs a compression and preevaluation of the files. Spot identification and quantification is performed by the chain code algorithm after appropriate zooming and cutting. Labeling facilitates spot identification and quantification in numerical and graphical (pseudocolor) representation on peripheral devices for camera ready output. Interpolation between measured basepoints is performed by cubic spline algorithms which are automatically switched on and off, depending on the need by the program. High speed analysis and graphic representation is achieved using fast Assembler language routines rather than high level languages. One‐dimensional gels can be analyzed using the same software. Spot matching between parallel two‐dimensional gels has not yet been implemented.