bluuebunny/crossref_metadata_2025_split huggingface dataset:
Two‐dimensional polyacrylamide gel electrophoresis reveals differences between osteoblast and fibroblast extracellular proteins
Authors: Hankey David P. , Nicholas R. M. , Hughes Anne E. 
Published: May 2005

AbstractNormal human skin fibroblast primary cell lines secrete over 50 proteins into culture medium. These have been mapped previously using two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) and this technique has now been used to investigate extracellular protein secretion by human osteoblasts in vitro. We report the mapping of a number of consistent markers specific to the osteoblast. In particular, one protein chain with posttranslational modifications was found to be unique to the osteoblast extracellular protein map. The absence of the N‐ and O‐glycoforms of collagenase from the osteoblast profile in this study concurs with findings reported using the immunoprecipitation functional assay and Northern blot analysis. The use of 2‐D PAGE in phenotypic assessment provides a more complete analysis than the standard range of single‐parameter tests for osteobiasts. Mapping of extracellular and cellular proteins in addition to bone matrix protein analysis will allow a comprehensive analysis of normal osteoblast function. This technique may also be applied to the study of osteoblasts in relation to bone disease and in assessing the phenotypic shift within a normal osteoblast culture.
Gel electrophoretic analysis of chitosan hydrolysis products
Authors: Audy Patrice , Asselin Alain 
Published: May 2005

AbstractEnzymatic hydrolysis of commercial crustacean chitosan by barley chitosanases was analyzed by subjecting chitosan to electrophoresis in a 10% w/v polyacrylamide slab gel in the presence of 7 M urea and 5.5% v/v acetic acid. Chitosan migrated as a polycation. Chitosan was stained with Coomassie Brilliant Blue R‐250 or visualized by ultraviolet transillumination after staining with Calcofluor White M2R. Some chitosan molecules were retarded by gel electrophoresis while small chitosan molecules migrated at the bottom of a 10% w/v polyacrylamide gel. Such analysis revealed that 96 h were necessary to convert all chitosan to oligosaccharides under our assay conditions. Chitosan oligosaccharides generated by enzymatic or chemical hydrolysis were further analyzed by electrophoresis in a 33% w/v polyacrylamide gel containing urea and acetic acid. Coomassie Brilliant Blue R‐250 was found to be better than Calcofluor White M2R for staining chitosan oligosaccharides. Chitosan oligomers of four residues (tetramers) or more were easily resolved in such a polyacrylamide gel system. To our knowledge, this is the first report of a gel electrophoretic separation of chitosan and its oligosaccharides.
Analysis of carbohydrate‐mediated heterogeneity and characterization of N‐linked oligosaccharides of glycoproteins by high performance capillary electrophoresis
Authors: Taverna Myriam , Baillet Arlette , Biou Daniel , Schlüter Michael , Werner Rolf , Ferrier Danielle 
Published: May 2005

AbstractCapillary zone electrophoresis (CZE) has been investigated as an alternative method to analyze the carbohydrate moieties of glycoproteins. Carbohydrate‐mediated microheterogeneity of the recombinant plasminogen activator (rt‐PA) was examined. The glycoprotein was resolved in multiple electrophoretic species using CZE but the separation was complicated by adsorption of the molecules to the wall of the capillary. The influence of several parameters, such as pH, molarity of the buffer and addition of a cationic additive, on the separation of glycopeptides was investigated. High resolution and reproducible separations of rt‐PA glycopeptides carrying hybrid and complex type chains were obtained using either a 100 mM phosphate buffer, pH 6.6, or a 100 mM Tricine buffer, pH 8.2, containing 1.25 mM of putrescine. N‐Oligosaccharides from fetuin, t‐PA and α1‐acid glycoprotein were separated within 20 min on the basis of both their sialic acid content and their structure. The use of an oligosaccharide fingerprinting technique, such as the present one, could have many applications in biotechnology to assess, for example, the consistency of production of a glycoprotein or for analytical glycoprotein chemistry.
Specific polypeptide markers in chronic B cell malignancies detected by two‐dimensional gel electrophoresis
Authors: Talebian‐Ziai Simin , Azgui Zahia , Valensi Françoise , Sigaux François , Charron Dominique 
Published: May 2005

AbstractTwo‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) analysis of silver stained polypeptides was used to discriminate the cell protein profiles in chronic B cell malignancies. We identified 5 distinct polypeptides (H1 to H5) present in hairy cell leukemia (HCL) (14 cases) and absent in most chronic lymphocytic leukemia (CLL) and normal B lymphocytes. Two polypeptides, H2 and H5, were absolutely specific of HCL. The 2‐D PAGE profiles found in 3 splenic lymphoma with villous lymphocytes (SLVL) and 2 HCL variants (HCL‐V) were similar to HCL with a unique additional peptide present in 3/3 SLVL and 1/2 HCL‐V. When HCL and SLVL were assessed for the CLL stage‐specific 2‐D PAGE patterns, HCL was related to stage A/B while SLVL was related to stage C. In conclusion, 2‐D PAGE analysis of the molecular pattern of B lymphocytes provides a new means to recognize the leukemic origin of the sample and distinguish HCL from CLL and SLVL.
A new technique for detecting oxytocinase activity in electrophoresis gels
Authors: Roy Ashim C. , Saha Nilmani , Tan Suan M. , Kamarul Farida Z. , Ratnam Shan S. 
Published: May 2005

AbstractA sensitive staining method for the detection of oxytocinase (EC 3.4.11.3) activity in electrophoresis gels has been described. The method is based on the enzymatic release of p‐nitroaniline (PNA) from two specific synthetic oxytocinase sub‐strates, S‐benzyl‐L‐cysteine‐p‐nitroanilide (BCN) and L‐leucine‐p‐nitroanilide (LN), respectively. The PNA is then diazotized with sodium nitrite and subsequently coupled to a chromogen, N‐(1‐naphtyl)‐ethylenediamine dihydrochloride (NED) to produce a deep pink/magenta colored azo‐dye at the site of oxytocinase activity.
Tissue‐specific pattern of nonhistone high mobility group proteins in various organs of the chicken
Authors: Pedrini Michael , Grunicke Hans , Csordas Adam 
Published: May 2005

AbstractElectrophoretic analysis of tissue‐specific differences of nonhistone high mobility group (HMG) proteins from nuclei of various organs of the chicken revealed that in organs with a higher proportion of replicating cells (thymus, Bursa Fabricii, spleen) the relative amount of HMG‐17 is considerably higher than that of HMG‐14; however, in transcriptionally active organs with a very small proportion of replicating cells (glandular stomach, liver) HMG‐14 and HMG‐17 are present at roughly equal and low amounts. In glandular stomach, liver and spleen, the relative contents of both HMG‐1 and HMG‐2 are markedly lower than in thymus and Bursa Fabricii. Moreover, the total amount of HMG proteins is higher in those organs which contain replicating lymphocytes.
Distinction between supercoiled and linear DNA in transverse agarose pore gradient gel electrophoresis
Authors: Wheeler David , Lin Jia‐Hwei , Chrambach Andreas 
Published: May 2005

AbstractFour species of linear DNA and the first four members of a linking series, generated by treatment of plasmid DNA (PUC19, 2.7 kb) with mitochondrial topoisomerase, were differentiated by transverse agarose pore gradient gel electrophoresis. The experimental curves of migration distance vs. agarose concentration (Ferguson curves) of supercoiled DNA exhibit a steeper trajectory than those of linear DNA of the same size range. As a consequence, the four supercoiled species exhibit an increase in apparent size (relative to linear DNA standards) with increasing agarose concentration. Both the crossing of the Ferguson curves with those of linear standards as well as the apparent size increase with agarose concentration can serve to detect supercoiled plasmid‐sized DNA in mixtures with linear DNA.
Rapid high resolution two‐dimensional electrophoresis of human sperm proteins
Authors: Kritsas John J. , Schopperle William M. , De Wolf William C. , Morgentaler Abraham 
Published: May 2005

AbstractAn improved protocol has been developed for two‐dimensional gel analysis of human sperm proteins through the application of recent technical advances. Advantages of this protocol consist of increased resolving power, reduced processing time, enhanced reproducibility of staining patterns, and applicability to small quantities of protein. Technical improvements include determination of optimal protein loading (20 micrograms/gel), development of a reliable tube gel casting system, and application of minigel technology. In this study over 500 proteins were resolved with molecular weights ranging from 12 000 to 105 000 and isoelectric points from 5.0 to 8.5. This is a three‐fold improvement in resolution over earlier results. A single operator was able to generate two‐dimensional gels of multiple samples in less than one day. This rapid high resolution technique should facilitate further investigation of human sperm proteins under normal and pathologic conditions.
Two‐dimensional electrophoresis of cat sera: Protein identification by cross reacting antibodies against human serum proteins
Authors: Miller Ingrid , Gemeiner Manfred 
Published: May 2005

AbstractAfter two‐dimensional electrophoresis of cat serum, individual proteins were identified by means of cross reacting antibodies against the human protein homologues. Testing by cross reactivities seemed the method of choice because immunoreagents for cat proteins are not easily available.
Using Coomassie Blue to stabilize H2O2‐guaiacol stained peroxidases on polyacrylamide gels
Authors: Fieldes Mary Ann 
Published: May 2005

AbstractShimoni and Reuveni [1] have reported a procedure which uses Coomassie Brilliant Blue R‐250 staining and destaining to stabilize H2O2‐guaiacol stained peroxidases on polyacrylamide gels and at the same time detects nonspecific protein bands. The procedure has the advantage that the otherwise orange and unstable peroxidase bands are visualized as blue bands and it, therefore, facilitates laser densitometry of electrophoretically separated peroxidases. In the modification of the procedure reported here, the Coomassie staining reagent was adjusted and the destaining step was eliminated. The modified procedure has added advantages: it simplifies the procedure, increases the consistency of the results across gels prevents or reduces the staining of nonspecific proteins, and still gives excellent resolution of stable bands which can be used to quantify peroxidase activity.
Separation of cresols using coelectroosmotic capillary electrophoresis
Authors: Masselter Sonja Marie , Zemann Andreas Josef , Bobleter Ortwin 
Published: May 2005

AbstractA new method of capillary electrophoresis was established to separate the three cresol isomers. Cresols are of importance due to their use as insecticides, precursors of various dye intermediates, resins, fire‐resistant fluids and other purposes. Reversion of the electroosmotic flow was carried out by a polycationic ammonium compound. The separations were carried out at high pH values (pH 12) to obtain complete dissociation of the cresols and short times of analysis (less than 5 min) through fast migration of the cresol towards the anode. Various aliphatic alcohols were added to improve peak shape and resolution.
Characterization of the electrophoretic properties of nucleosome core particles by transverse polyacrylamide pore gradient gel electrophoresis
Authors: Orbán Lászlo , Garner Mark M. , Wheeler David , Tietz Dietmar , Chrambach Andreas 
Published: May 2005

AbstractTransverse pore gradient gel electrophoresis, previously applied to bent DNA, has extended the usefulness of the gel retardation assay in two ways: (i) by differentiating between different DNA conformations; (ii) by providing information regarding the physical properties of DNA. In the present study, similarly extended information is obtained with regard to a well‐characterized DNA‐protein complex, the chicken erythrocyte nucleosome core particle. (i) The winding of DNA around the protein core constrains the DNA which renders its Ferguson curve (migration distance vs. gel concentration) similar to that of kinetoplast DNA, i.e. it intersects sharply with the Ferguson curves of linear DNA standards. By contrast, the deproteinized nucleosome DNA exhibits a Ferguson curve similar to linear standards of the same lenght. (ii) Interpretation of the Ferguson curve based on a mathematical model shows that the nucleosome exhibits a linear Ferguson plot [log(mobility) vs. gel concentration]. This is similar to and characteristic of spherical proteins, contrasting with the concave plot typical for linear and bent DNA. (iii) The effective size of the nucleosome, evaluated in terms of an “equivalent sphere” (i.e. a hypothetical spherical particle with a radius, Res, having the same electrophoretic mobility as DNA for a particular set of experimental conditions), remains invariant across the gel concentration range of 3–9 % T. This is similar to proteins and bacteriophages and contrasts with the progressive decline of Res with increasing gel concentration observed for linear DNA and the deproteinized nucleosomal DNA.
Analysis of plasmid‐DNA and cell protein of recombinant Escherichia coli using capillary gel electrophoresis
Authors: Hebenbrock Kirstin , Schügerl Karl , Freitag Ruth 
Published: May 2005

AbstractPlasmid DNA prepared from cultivation samples of recombinant Escherichia coli was analyzed by capillary gel electrophoresis. We used this method to control the genetic stability during a fed‐batch culture. The plasmid DNA and a standard DNA mixture with molecular weights in the size range of 3000–22 000 base pairs (bp) were analyzed in capillaries that were filled with solutions of non‐cross‐linked polyacrylamide. With this method the plasmid DNA from recombinant E. coli cultivation samples was analyzed within 30 min. Separation parameters such as gel concentration, capillary length and current were optimized for that purpose. The method was modified to allow the analysis of proteins. Crude cell lysate samples could be screened for the protein pattern within 15 min in sodium dodecyl sulfate‐polyacrylamide filled capillaries. The method was also used to verify product purity after the down‐stream process.
A traveling‐wave micropump for aqueous solutions: Comparison of 1 g and μg results
Authors: Müller Torsten , Arnold W. Michael , Schnelle Thomas , Hagedorn Rolf , Fuhr Günter , Zimmermann Ulrich 
Published: May 2005

AbstractThe operation of a micro‐chip‐based fluid circulator based on the interaction of a high‐frequency traveling wave with a thermally generated inhomogeneity in an aqueous medium is investigated. The profiles of the electric field, the thermal gradient and the driving force within the device are derived numerically, and the dependence upon applied voltage under different experimental conditions is derived. The importance of convection in the operation of the device is assessed by operating it in various orientations, as well as under microgravity, and also by investigating the reversal and turn‐on characteristics. The pump showed the unexpected ability to trap microparticles present in the driven fluid.
Capillary electrophoresis quantitation of l–L–folinic acid in the presence of its inactive d–L–form
Authors: Cellai Luciano , Desiderio Claudia , Filippetti Raffaella , Fanali Salvatore 
Published: May 2005

AbstractThe diastereomers d‐L and l‐L‐folinic acid were separated by capillary electrophoresis, using heptakis (2,3‐di‐O‐methyl)‐β‐cyclodextrin as a chiral active component in the background electrolyte. The method proved suitable for the quantitation of these compounds in one commercial pharmaceutical preparation.
Simultaneous determination of native and subunit molecular weights of proteins by pore limit electrophoresis and restricted use of sodium dodecyl sulfate
Authors: Tyagi Rakesh Kumar , Babu Batchu Ramesh , Datta Kasturi 
Published: May 2005

AbstractIn the present communication, we describe a method for the determination of both the native and subunit molecular weight of a protein by a single pore limit electrophoretic run. When native proteins and sodium dodecyl sulfate (SDS)‐heat denatured proteins were electrophoresed in a 4–28% gradient‐polyacrylamide gel, in the presence of 0.02% SDS in running buffer, their respective molecular weights (native and subunit) could be determined by comparing the migration distances of unknown and standard proteins. The presence of SDS up to 0.02% in the running buffer did not dissociate the native proteins into their subunits. The linearity in distribution of native and SDS‐polyacrylamide gel electrophoresis (PAGE) standard marker proteins used for the molecular weight determination further showed the efficacy of the present technique.
Lack of inter‐species reactivity between antigens and antibodies is overcome by protease treatment of Western blots
Authors: Pan Jie , Pfeiffer David C. , Reimer Corinne L. , Crawford Bruce J. , Vogl A. Wayne , Auersperg Nelly 
Published: May 2005

AbstractThe analysis of proteins by Western blotting is frequently limited by the inability of antibodies to recognize antigenic epitopes in proteins of different species. In the present study, we investigated the influence of mild protease digestion on the reactivity of nitrocellulose‐blotted proteins and microscopic sections with antibodies produced against analogous proteins of different species. The proteins were partially purified, electrophoresed and blotted by standard procedures. They were then incubated with either trypsin or pepsin, at concentrations ranging from 0.5 to 80 μg/mL, for different time periods prior to reaction with the first antibody. After incubation with the second antibody, the bands were visualized by chemiluminescence. The following antibody/antigen pairs produced no signal by conventional methods but resulted in the appropriate bands following protease treatment: (i) monoclonal antibody (Mab) to human keratin #18 / rat keratin; (ii) Mab to starfish extracellular matrix/ mouse laminin; (iii) rabbit antiserum to human platelet myosin II/ratfish nonmuscle myosin II. In the latter system, protease treatment also revealed previously hidden epitopes in microscopic sections. Appropriate controls demonstrated that the antibodies retained their specificity after the protease treatment of the preparations. Optimal conditions varied and had to be defined for each protein under study. The results suggest that protease treatment of immunoblots reduces the lack of inter‐species reactivity between antibodies and antigens.
Immunoaffinity analysis of cross‐reacting allergens by protein blotting
Authors: Donovan Gregory R. , Baldo Brian A. 
Published: May 2005

AbstractIgE antibodies from sera having reactivity against ryegrass pollen protein allergens, wheat endosperm protein allergens and also several other cereal protein allergens were adsorbed with either ryegrass pollen or the wheat/globulin fraction immobilised on solid phases and subsequently eluted with low pH buffer. The eluted antibodies were reacted with sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) blots of the different allergens. Antibodies adsorbed and subsequently eluted from the two allergen sources recognised different spectra of proteins in the ryegrass pollen and cereal allergen sources and indicated the degree of immunological cross‐reactivity. Intra‐species cross‐reactivity of IgE antibodies was demonstrated employing similar methods to those used for the pollen and cereal allergens by using a recombinant allergen from the venom of the ant Myrmecia pilosula as the immunoadsorbant protein on the solid phase.
Calculating paternity probabilities from DNA multilocus fingerprints in some cases of deficiency
Authors: Yassouridis Alexander , Epplen Jörg T. 
Published: May 2005

AbstractPaternity determination via DNA multilocus fingerprints in normal trio cases, namely when the fingerprints of the trio mother, child and putative father are available, is performed on a routine basis. But in cases of deficiency, where the DNA fingerprint of mother or putative father is missing, there exists no systematic, analytical way for paternity calculations, even in cases where DNA fingerprints of other persons related to the missing individual are available. The aim of this paper is to establish a mathematical, analytical background, that enables paternity calculations in cases of deficiency if complementary information can be obtained from the DNA fingerprints of at least one of the parents of the missing individual. Using the basic outline of our previously described procedure, the formulas for paternity calculations also allow mutations.
The fractional volume available to prolate spheroids in a network of randomly oriented fibers obtained by computer modeling: Correlation with the Ogston equation
Authors: Wheeler David , Chrambach Andreas 
Published: May 2005

AbstractComputer modeling was used to measure the fractional volumes available to prolate spheroid objects in a random, inert network of fibers. The data fit the Ogston equation exactly when the object was a sphere (axial ratio = 1). When the axial ratio was increased from 1 to 9, the Ogston equation was still obeyed if the fiber concentration is multiplied by a factor, A, which increases linearly in proportion to the axial ratio. The factor A allows one to adjust the retardation coefficient derived from gel electrophoresis, KR, for spherical objects to that of prolate spheroids with axial ratios from 1 to 9. Potentially, the same adjustment of KR is possible for objects of other shapes.
Isotachophoresis of proteins in sucrose density gradients
Authors: Acevedo Fernando 
Published: May 2005

AbstractThe separation of proteins from human serum by isotachophoresis in sucrose density gradients, with mixtures of discrete amphoteric substances as spacers, is described. Open columns and columns with a dialysis membrane to hold the sucrose gradients were used. A simple algorithm based on the Kohlrausch function was used to calculate the amount of each spacer. The pH gradients generated in open columns were found to be in agreement with the calculations. The load was up to two gram proteins. The analysis of the fractions obtained after the separation showed a distribution of components similar to as analytical isotachophoresis. It is concluded that sucrose density gradients are suitable as supporting media for the preparative separation of proteins by isotachophoresis. The high resolution attained and the possibility of scaling‐up the separation systems are major advantages of this system. In addition, the sample is easily and completely recoverable.