bluuebunny/crossref_metadata_2025_split huggingface dataset:
Protein blotting followed by microsequencing
Authors: Choli Theeodora , Wittmann‐Liebold Brigitte 
Published: May 2005

AbstractThe use of new membranes such as activated or derivatized glass fibers as well as synthetic membranes, which are compatible with the hazardous sequencing reagents, are described. Precautions to be taken in order to prevent N‐terminal block age of the proteins during electrophoresis and blotting are described, as well as the conditions for protein detection after blotting and protein treatment for in situ amino acid analysis, fragmentation and microsequencing. For a number of standard proteins and bacterial ribosomal proteins microsequence analysis is reported for two commercially available sequencers (Applied Biosystems and Knauer).
A gamma‐ray detector for capillary zone electrophoresis and its use in the analysis of some radiopharmaceuticals
Authors: Altria K. D. , Simpson Colin F. , Bharij A. K. , Theobald A. E. 
Published: May 2005

AbstractThe construction and evaluation of a gamma‐ray detector for capillary zone electrophoresis is described. The detector was shown to have a linear response from the limit of detection 10 to 500 Bqcm−3 corresponding to 5.1 x 10−17 to 2.55 x 10−15 gcm−3 43Tc99m. The application of the detector for the analysis of some radiopharmaceuticals is presented.
Applications of capillary electrophoresis for industrial analysis
Authors: Stover Frederick S. 
Published: May 2005

AbstractCapillary electrophoresis techniques, particularly isotachophoresis and zone electrophoresis, are useful for the determination of various analytes in a wide range of sample matrices. This paper reports applications of capillary electrophoresis developed for detergent, food additive, herbicide, animal nutrition and biotechnology samples. Major advantages of capillary electrophoresis include versatility and speed of method development. Options for small ion analysis using modern, fused silica capillary instruments are discussed.
Enantiomer resolution by using capillary zone electrophoresis: Resolution of racemic tryptophan and determination of the enantiomer composition of commercial pharmaceutical epinephrine
Authors: Fanali Salvatore , Boček Petr 
Published: May 2005

AbstractThe enantiomers of D‐ and L‐tryptophan were separated by capillary electrophoresis, using α‐cyclodextrin as a chiral active component in the background electrolyte. The separation of (‐) and (+) epinephrine was achieved by supplementing the background electrolyte with Heptakis (2,6‐di‐O‐methyl‐β‐cyclodextrin). As a practical application of the method, the quantitative analysis of (‐)and(+)enantiomers in commercial pharmaceutical solutions of adrenaline is shown.
Capillary electrophoretic determination of S‐carboxymethyl‐L‐cysteine and its major metabolites in human urine: Feasibility investigation using on‐column detection of non‐derivatized solutes in capillaries with minimal electroosmosis
Authors: Tanaka Yoshio , Thormann Wolfgang 
Published: May 2005

AbstractThe capillary electrophoretic analysis of S‐carboxymethyl‐L‐cysteine and some of its metabolites in human urine is reported using (i) on‐column detection of underivatized solutes, (ii) minimal sample pretreatment and (iii) capillary columns with minimized electroosmosis. Experimental results obtained with two apparatus, the HPE‐100 (Bio‐Rad), featuring coated fused silica capillaries of 25 μm i.d., and with the Tachophor 2127 (LKB), having Teflon capillaries of 500 μm i.d. are discussed. Drug concentrations down to 0.2 mg/mL on capillary isotachophoresis and 0.03 mg/mL on capillary zone electrophoresis can be monitored with these instruments.
The use of metalion‐supplemented buffers to enhance the resolution of peptides in capillary zone electrophoresis
Authors: Mosher Richard A. 
Published: May 2005

AbstractThe potential of metal ion‐containing buffers to enhance the resolution of peptides in capillary zone electrophoresis was evaluted. The impact of adding Cu(II) and Zn(II) salts to electrophoresis buffers is shown to affect the migrational behavior of several dipeptides containing histidine. Interaction with a metal ion differentially decreases the electrophoretic mobilities of peptides which comigrate in the absence of metal ions, thus causing their separation. This effect is obtained at low pH where the large net charge on the samples yields short analysis times. The dependence of the resolution on Zn(II) concentration is presented for two different samples. The influence of the background buffer is discussed.
The effects of electroosmosis on the structure of isotachophoresis boundaries
Authors: Saville Dudley A. 
Published: May 2005

AbstractThe influence of convection on the structure of an isotachophoretic boundary is investigated using an asymptotic method to simplify the conservation equations. To illustrate the methodology it is applied to a simple strong electrolyte system consisting of three ions and the relevant equations solved numerically. The technique, however, is of general applicability and can be useful in exploring the influence of various flow processes on analytical and preparative electrophoretic separation techniques in addition to isotachophoresis.
Comparative studies of recycling isoelectric focusing and continuous flow electrophoresis: Separation of proteins with minor charge differences
Authors: Knisley Keith A. , Rodkey L. Scott 
Published: May 2005

AbstractContinous flow zone electrophoresis (CFE) and recycling isoelectric focusing (RIEF) are two of the alternative formats for fluid phase preparative isolation of biological products in liquid separation media. The McDonnell Douglas CFE system has been used for both ground‐based and microgravity separations. The ground‐based McDonnell Douglas CFE and RIEF were compared for the ability to reslove mixtures of proteins with known charge differences. Mixtures of (1) cytochrome c, myoglobin, and ovalbumin or (2) beta‐lactoglobulin and ovalbumin were used to evaluate the resolving capabilities of CFE and RIEF. Following separation, fractions were analyzed by determining absorbance at 280 nm and by analytical isoelectric focusing (IEF) using Coomassie Brilliant Blue or silver staining to detect focused proteins. Both CFE and RIEF apparently separated the components of both mixtures into individual peaks, separated by fractions which contained little or no detectable protein. Coomassie‐stained analytical IEF gels supported this finding. However, when separated proteins were analyzed by silver staining of the analytical gels, the separation of ovalbumin from beta‐lactoglobulin by CFE was not complete. Ovalbumin was free ofbeta‐lactoglobulin but beta‐lactoglobulin was contaminated by trace amounts of ovalbumin. RIEF clearly separated each protein with no detectable contamination. These data demonstrate the superiority of RIEF over CFE for resolution of protein mixtures having only minor charge differences. RIEF may be more efficient due to the documented electrodissociation of noncovalent protein:protein complexes which occurs during RIEF separations.
Correlation of capillary zone electrophoresis with continuous free‐flow zone electrophoresis: Application to the analysis and purification of synthetic growth hormone releasing peptide
Authors: Prusik Zdeněk , Kašička Václav , Mudra Petr , Štěpánek Jiří , Smékal Otto , Hlaváček Jan 
Published: May 2005

AbstractTwo carrier‐free electrophoretic separation methods, capillary zone electrophoresis (CZE) and continuous free‐flow zone electrophoresis (FFZE), have been applied to both microanalysis at the nanogram level and preparative fractionation, with a throughput of 30 mg/h, of synthetic growth hormone releasing peptide (GHRP). A crude product of GHRP, a hexapeptide with the sequence His‐D‐Trp‐Ala‐Trp‐D‐Phe‐Lys‐NH2, synthesized by the solid phase methodology, was desalted and analyzed by CZE. Based on the results of analytical CZE the separation was converted into a preparative purification procedure by continuous FFZE, employing the same separation medium (0.5 mol/L acetic acid, pH 2.6). The purity of peptide frcttions obtained by FFZE was reevaluated by CZE. The combination of these two techniques proved to be a valuable tool for both peptide analysis and peptide purification. A close correalation of CZE and FFZE, resulting from the fact that both methods are based on the same separation principle (zone electrophoresis) and that both are performed in a free solution of the same composition, was confirmed. However, when transforming data from CZE to FFZE, the different electroosmotic flow, temperature and electric field intensity in the capillary and in the flow‐through cell, respectively, have to be taken into account and corresponding corrections have to be made.
Separation of enzymes from Candida boidinii crude extract by continuous flow zone electrophoresis
Authors: Nath Sunil , Schütte Horst , Weber Gerhard , Hustedt Helmut , Deckwer Wolf‐Dieter 
Published: May 2005

AbstractSeparation of the enzymes formate dehydrogenase, formaldehyde dehydrogenase and methanol oxidase from Candida boidinii crude extract has been explored using continuous flow zone electrophoresis in the VaP‐22 and the scaled‐up VaP‐220 electrophoresis apparatus. Yields up to 95 % and purification factors between 3 and 7 were obtained, together with separation of cell debris from the enzymes. Multiple injections of sample were used to obtain a protein throughput of 46.2 mg/h in the VaP‐22. A tenfold higher throughput was achieved using the VaP‐220. Correlation of the electrophoretic mobility in continuous flow zone electrophoresis with the elution behavior in ion‐exchange chromatography confirmed the primary role of net surface charge in the separation of biological molecules. Proteins and enzymes with differences > 0.05 M elution molarities in ion‐exchange chromatography can be separated. This corresponds to a preparative scale (mg/h or g/h) separation of proteins and enzymes whose difference in apparent electrophoretic mobility is > 0.70 x 10−5cm2/(V.s).
Nonamphoteric isoelectric focusing: II. Stability of borate‐glycerol pH gradients in recycling isoelectric focusing
Authors: Todd Paul , Elsasser William 
Published: May 2005

AbstractBy complexing polyols with borate in recycling isoelectric focusing and by varying the ratio of polyol to borate over the useful pH range of 4.0‐6.0, it is possible to control pH. Twelve solutions of 0.1 M boric acid and varying glycerol concentration were used to vary pH in a twelve‐compartment commercial recycling isoelectric focusing (RIEF) system. Various concentrations of boric acid were tested as anolyte, and various Tris(hydroxymethylamino)methane‐borate buffer systems were tested as catholyte. Electroosmosis, hydrogen ion flow, and fluid balancing were characterized in two glycerol gradients; one was maintained at 0.06 pH/fraction and the other at 0.12 pH/fraction. In the latter case, ovalbumin (pI 4.70) migrated to the pH 4.61 and 4.72 compartments. It is concluded that the borate‐glycerol system can be adequately stabilized in RIEF for isoelectric purification of certain proteins.
Shifts of isoelectric points between cellular and secreted antibodies as revealed by isoelectric focusing and immobilized pH gradients
Authors: Wenisch Elisabeth , Reiter Susanne , Hinger Susanne , Steindl Franz , Tauer Christa , Jungbauer Alois , Katinger Hermann , Righetti Pier Giorgio 
Published: May 2005

AbstractCharge microheterogeneity of monoclonal antibodies, as revealed by isoelectric focusing in carrier ampholytes, has been known for a long time. Here we demonstrate, in the case of monoclonals against the gp‐41 of the HIV‐1 virus, that this heterogeneity is already present within the cell sap of hybridoma cells during antibody synthesis. When the monoclonals are secreted extracellularly, the same isoelectric point (pI) spectrum is maintained, but there is a marked redistribution of the relative isoform abundance towards the lower pI components. This suggests in vivo processing of such forms, possibly via glycosylation or deamidation. The secreted antibodies are also analyzed by immobilized pH gradients (IPG), where they demonstrate an even more extensive heterogeneity, due to the marked increment in resolving power. Single bands are purified by preparative IPGs in a multicompartment electrolyzer and are shown to be stable with time. Thus, artefactual heterogeneity produced by the focusing technique is completely excluded and cellular processing is clearly established.
Effects of methanol and urea on optical resolution of phenylthiohydantion‐DL‐amino acids by micellar electrokinetic chromatography with sodium N‐dodecanoyl‐L‐valinate
Authors: Otsuka Koji , Terabe Shigeru 
Published: May 2005

AbstractEffects of methanol and urea on enantiomeric resolution of phenylthiohydantoin (PTH)‐DL‐amino acids by micellar electrokinetic chromatography with sodium N‐dodecanoyl‐L‐valinate (SDVal) were investigated. PTH‐DL‐Met that could not be optically resolved with an SDVal solution alone was resolved by using an SDVal/methanol solution. Four PTH‐DL‐amino acids were separated from each other and each pair of enantiomers was also optically resolved, although seriously tailed peaks were observed for each solute. The addition of urea to SDVal/methanol solutions improved these peak shapes.
Imaging as a tool for improving length and accuracy of sequence analysis in automated fluorescence‐based DNA sequencing
Authors: Sanders Jane Z. , Petterson A. Albert , Hughes Peter J. , Connell Charles R. , Raff Malcolm , Menchen Steven , Hood Leroy E. , Teplow David B. 
Published: May 2005

AbstractA new method of signal analysis for automated fluorescence‐based DNA sequencing is presented. Signal resolution is a limiting factor in obtaining accurate sequence information beyond 400–450 nucleotides per gel lane. We have developed a computer program for the imaging of DNA bands in sequencing gels. The image analysis shows that distortions in the shapes of the bands decrease resolution of peaks observed in the standard data plots. Reconstruction of the undistorted band shape prior to signal analysis substantially improves the resolution of peaks and may improve the accuracy and length of the contiguous sequence read. Image analysis identified other factors limiting the accuracy and length of automated DNA sequence analysis and provided a tool for evaluating various remedies. Our techniques should also be applicable in other systems, for example, in gel electrophoresis of proteins and DNA restriction fragments, and in scranning densitometry.
Analysis of one‐dimensional gels and two‐dimensional Serwer‐type gels on the basis of the extended Ogston model using personal computers
Authors: Tietz Dietmar 
Published: May 2005

AbstractThis reportPreliminary reports of this study were presented at the American Electrophoresis Society Meeting, Washington DC, USA, July 6–11, 1989; at the NIH/ADAMHA‐Industry Collaboration Forum, Bethesda, Maryland, USA, October 3, 1989; at the Electrophoresis Forum '89, Munich, Germany, October 23–25, 1989; and at Frontiers in Bioprocessing II, Boulder, Colorado USA, June 17–21, 1990.
presents the stand‐alone computer application ELPHOFIT, a software package for the analysis of gel electrophoretic data based on Ferguson plots. Either conventional one‐dimensional gels or two‐dimensional agarose gels (Serwer‐type) can be evaluated. Special emphasis is on the latter gel type, which has been applied previously for the separation of DNA, intact viruses and polydisperse meningitis vaccines. ELPHOFIT is designed for Macintosh PCs and for the IBM XT, AT, PS/2 and compatibles. The program operates interactively with the user, who determines the course of evaluation. Data input is in the format of files providing values of gel electrophoretic migration distances or particle mobility (absolute or relative). Data processing involves a simultaneous least‐square curve fitting algorithm (Newton‐Gauss, Marquardt‐Levenberg) which uses equations derived from the extended Ogston model. Functions are fit to the database by adjusting their variables, representing physical parameters of the gel and the electrophoresed particle. The program output consists of tables and graphics accompanied by an explanatory text providing the following information: (i) radius and free mobility of the electrophoresed particle, (ii) fiber radius, length and volume, mean or median pore radius of the gel, (iii) linear Ferguson plots, (iv) iso‐free‐mobility/iso‐size nomogram for two‐dimensional gels, (v) confidence ellipses, (vi) required parameters or image processing program GELFIT and (vii) goodness‐of‐fit and other statistical parameters, such as standard errors, dependency values, root‐mean‐square (RMS) error and determination coefficient. Other features of the program are (i) simulation of Serwer‐type two‐dimensional electrophoresis, (ii) standardization according to size, or size and free mobility, (iii) the conversion of particle radii to molecular (or particle) weight and vice versa, (iv) interconversion of DNA size specifications, i.e. the number of base pairs and the geometric mean radii, (v) computation of gel concentration for optimal resolution of two components, (vi) option to obtain a session record, (viii) option to establish a data output file containing the information of generated graphics (IBM only) and (ix) a text editing facility, e.g., for creating data files. Graphics (Macintosh version, PICT format) and text output files (both IBM and Macintosh versions, standard ASCII format) generated by ELPHOFIT are compatible with commercially availabe software.
Phenotyping of apolipoprotein E by immunoblotting in immobilized pH gradients
Authors: März Winfried , Cezanne Sabine , Groß Werner 
Published: May 2005

AbstractAn immunoblotting method for the determination of apolipoprotein E (apoE) phenotypes has been developed. Delipidated plasma proteins are focused in an immobilized pH gradient, and transferred to polyvinylidene difluoride (PVDF) membranes. ApoE isomorphs are identified by immunoperoxidase staining. The method allows reproducible assignment of apoE phenotypes without isolation of triglyceride‐rich lipoproteins. Only small amounts of serum are required. There are several important steps in the procedure: (i) delipidation is indispensable, (ii) carrier ampholytes have to be added to the gels and to the sample buffer, and, (iii) on immunostaining, polyvinylidene difluoride membrane provide an excellent signal‐to‐background ratio.
Resolving isoforms of aldose reductase by preparative isoelectric focusing in the Rotofor
Authors: Petrash J. Mark , Delucas Lawrence J. , Bowling Earnest , Egen Ned 
Published: May 2005

AbstractWe have resolved and characterized isoforms of aldose reductase from bovine and porcine lenses by preparative isoelectric focusing with narrow pH gradients using the Rotofor. Both bovine and porcine lens aldose reductases were resolved as two enzyme isoforms. The bovine isoforms were Mr40400 ± 445 polypeptides of pI 4.71 and 5.19. Porcine isoforms were Mr 41500 ± 450 polypeptides of pI 4.90 and 5.30 Staphylococcus aureus V‐8 protease digestion patterns for each set of isoforms were essentially identical and all isoforms probably contain blocked amino terminal amino acids. Antiserum to bovine lens aldose reductase cross‐reacted with porcine lens aldose reductase. Each isoform displayed substrate preferences characteristic of mammalian aldose reductases. With purification, both bovine and porcine lens aldose reductases became less senstive to inhibition by 6‐fluoro‐spiro‐(chroman, 4,4′‐imidazolidine)‐2′,5′‐dione (sorbinil).
The grammatical rule for all DNA: Junk and coding sequences
Authors: Ohno Susumu , Yomo Tetsuya 
Published: May 2005

AbstractSelfish DNA, coding sequences, and junk DNA in the genome are no stranger to each other; rather, they represent three phases in the life cycle of DNA. Accordingly, they all obey the same grammatical rule of TG/CA/CT excess and CG/TA deficiency. On the one hand, it is this very rule which keeps isoelectric points of most proteins near the neutral range. On the other hand, this rule creates numerous palindromes, thus maintaining symmetry between complementary strands. Many of these palindromes encode identical oligopeptides on both strands.
Isolation of simple‐sequence loci for use in polymerase chain reaction‐based DNA fingerprinting
Authors: Rassmann Kornelia , Schlötterer Christian , Tautz Diethard 
Published: May 2005

AbstractSimple sequences are short regions of tandem repetitions of mono‐, di‐, tri‐, or tetranucleotide motifs and occur as repetitive elements in all eukaryotic genomes. These regions tend to be hypervariable in length and can therefore be exploited for DNA fingerprinting purposes, using the polymerase chain reaction with primers flanking such regions. We describe how suitable simple‐sequence loci can be isolated from any given eukaryotic DNA. We show the DNA sequences for a number of variants of such loci and discuss the current results on their usefulness for DNA fingerprinting.
Detection of single and multiple polymorphic loci by synthetic tandem repeats of short oligonucleotides
Authors: Vergnaud Gilles , Mariat Denis , Zoroastro Monique , Lauthier Valérie 
Published: May 2005

AbstractLoci containing tandem repeats of short sequences are sometimes associated with a high level of polymorphism due to variations in the number of repeats. The different variants can be easily characterized by Southern blotting when the repeats span a range from a few hundred bases to a few kilobases, and probes derived from such tandem repeats constitute convenient genetic markers. These structures, usually called minisatellites, are best documented in the human genome, where their number has been estimated to be at least 1500. However, their role and mode of evolution are poorly understood. We are developing tools to evaluate the number of such redunddant sequences in a genome and to gain access to new polymorphic loci. Our strategy is based on the use of polymers of oligonucleotides as DNA probes for hybridization on Southern blots. In a previous report, we made polymers with random units of 14 bp and showed that they detect multiple polymorphic loci on human genomic DNA. At present, we are testing the effect of an increase in the complexity of the polymer, as obtained by the use of a longer random unit, and the effect of slight sequence modifications to a particular tandem repeat sequence. In addition, some of these synthetic probes can detect a single polymorphic locus and directly provide new genetic markers.
DNA bioprints: Simple nonisotopic DNA fingerprints with biotinylated probes
Authors: Pena Sérgio D. J. , Macedo Andrea M. , Gontijo Nelder F. , Medeiros Arnaldo M. , Ribeiro Julio Cesar C. 
Published: May 2005

AbstractThe discovery of multilocal DNA fingerprinting represented a revolution in criminal identification and paternity testing. However, for routine use in clinical laboratories, the standard DNA fingerprint methodology is too complex. We have been successful in the development of a simplified DNA nonisotopic fingerprinting system using biotin‐labeled probes which we have called DNA bioprints. To achieve this we explored three main technical features: utilization of biotinylated nonradioactive probes as a simpler substitute for 32P‐labeled probes, utilization of oligonucleotide probes as a simpler substitute for recombinant probes, and direct hybridization in the dried agarose gel as a simpler substitute for Southern blots. In this article we review our results in the development of DNA bioprints.
DNA fingerprinting in domestic animals
Authors: Buitkamp Johannes , Ammer Hubert , Geldermann Hermann 
Published: May 2005

AbstractDNAs of several species of domestic animals digested with the restriction endonucleases HinfI, AluI and HaeIII were hybridized with different synthetic probes. DNA fingerprint patterns were found in each investigated species by at least two of these probes. Furthermore, two probes gave sex‐specific banding patterns in the chicken. Some applications of DNA fingerprinting in domestic animals are discussed.