bluuebunny/crossref_metadata_2025_split huggingface dataset:
Microsequence analysis of the N‐terminally blocked proteins immobilized on polyvinylidene difluoride membrane by Western blotting
Authors: Hirano Hisashi , Komatsu Setsuko , Kajiwara Hideyuki , Takagi Yoshiaki , Tsunasawa Susumu 
Published: May 2005

AbstractA technique has been developed for efficient deblocking and subsequent microsequencing of N‐terminally blocked proteins immobilized on a polyvinylidene difluoride (PVDF) membrane at the picomole levels. In this technique, proteins were first separated by polyacrylamide gel electrophoresis and then transferred onto a PVDF membrane by Western blotting. The electro‐blotted proteins with N‐terminal acetylserine or acetylthreonine could be deblocked on‐membrane by treatment with trifluoroacetic acid vapor and sequenced by a gas‐phase protein sequencer. Similarly, N‐formylated proteins could be deblocked on‐membrane in HC1 solution and then directly sequenced from the N‐terminal amino acid. Proteins with N‐terminal pyroglutamic acid were enzymatically deblocked by in situ pyroglutamyl peptidase digestion, and N‐acetylated proteins were also enzymatically deblocked with acylamino acid‐releasing enzyme (AARE) after on‐membrane digestion with trypsin to generate the N‐terminal peptide fragment. This tryptic digestion was required since AARE can remove the acetylamino acid only from a short peptide. Based on these four deblocking methods, we present a strategy for sequential deblocking and subsequent N‐terminal sequence analysis of N‐blocked protein immobilized on PVDF membrane.
Time‐resolved fluorometry in nucleic acid hybridization and Western blotting techniques
Authors: Diamandis Eleftherios P. 
Published: May 2005

AbstractTime‐resolved fluorometry is now used extensively for immunological assays and to a lesser extent in other research areas. In this review I describe applications of time‐resolved fluorometry in nucleic acid hybridization and in blotting techniques, including Southern and Western blotting. Clearly, timeresolved fluorometry has potential for playing a major role in techniques other than immunological assays.
Protein blot assays specific for the discrimination of the centromere autoantigen, CENP‐A, from human cells
Authors: Billings Peter B. , Martinez Antigona , Haselby Jeanne A. , Hoch Sallie O. 
Published: May 2005

AbstractThe Western or protein blot has proven to be a valuable resource in detecting discrete, immunoreactive antigen targets associated with a variety of autoimmune diseases. As the roster of autoantigens has expanded, it has become increasingly common to tailor specific gel or blot conditions to a particular polypeptide antigen. Two such assays are reported here as applied to the fractionation and visualization of human centromere protein (CENP‐A), a centromere autoantigen associated with the rheumatic disease, systemic sclerosis. The centromere antigens are effectively solubilized in the presence of 1 M MgCl2 to allow for further purification. CENP‐A copurifies with the histone proteins, primarily H3 and H4. The two CENP‐A‐specific protein blot assays separate CENP‐A from the histone proteins and enhance CENP‐A immuno‐reactivity. The first assay is based on the use of acid‐urea gels with a Triton X‐100 concentration chosen to maximize separation of CENP‐A from all the histones. The second assay is based on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis to differentiate two very basic proteins of similar molecular weight, namely CENP‐A and histone H3. For each gel system, a selective choice of associated immunoblot parameters allows for the reproducible discrimination of the CENP‐A antigen.
The Pearson product‐moment correlation coefficient is better suited for identification of DNA fingerprint profiles than band matching algorithms
Authors: Häne Bernhard G. , Jäger Kathrin , Drexler Hans G. 
Published: May 2005

AbstractA database of DNA fingerprint profiles from permanently established human and animal cell lines was prepared with a computer program originally designed for numerical taxonomy of bacteria. Identifications of cell line DNA profiles were performed, both by the Pearson product‐moment correlation coefficient and by band matching. Under the conditions used the Pearson product‐moment correlation coefficient was consistently more reliable.
Heterogeneity of carbonic anhydrase and 68 kDa neurofilament in nerve roots analyzed by two‐dimensional electrophoresis
Authors: Fujii Hiroshi , Nakamura Kazuyuki , Takeo Kazusuke , Kawai Shinya 
Published: May 2005

AbstractThe heterogeneity of carbonic anhydrase (CA) and 68 kDa neurofilament (NF) in nerve roots of Japanese monkeys (Macaca Macacus) was analyzed by two‐dimensional electrophoresis combined with immunoblotting. CA was detected as heterogeneous spots of type I and II having the same molecular weight (Mr) but different isoelectric point (pI), in the range of pH 4.5–5.5. CA had much higher concentrations in soluble fractions of dorsal roots than ventral roots but not in membrane‐bound fractions of either root. NF was detected as a single spot at the position of 68 kDa and pI 5.2 in soluble fractions of both roots, however, it was detected in the membrane‐bound fractions as heterogeneous spots having different molecular masses and pI in the range of 60–68 kDa and 5.0–5.5, respectively. The presence of heterogeneous CA type I and II at high concentrations in dorsal roots enables us to distinguish the sensory nerves from the motor nerves. Furthermore, the heterogeneity of NF in membrane‐bound fractions gives us information for understanding the dynamic assembly and the degradation of NF in nerve roots.
The human keratinocyte two‐dimensional gel protein database: Update 1993
Authors: Celis Julio E. , Rasmussen Hanne Holm , Olsen Eydfinnur , Madsen Peder , Leffers Henrik , Honoré Bent , Dejgaard Kurt , Gromov Paul , Hoffmann Hans Jürgen , Nielsen Morten , Vassilev Alex , Vintermyr Olav , Hao Junshan , Celis Ariana , Basse Bodil , Lauridsen Jette B. , Ratz Gitte P. , Andersen Annette H. , Walbum Else , Kjærgaard Inge , Puype Magda , Van Damme Josef , Vandekerckhove Joël 
Published: May 2005

AbstractThe master two‐dimensional gel database of human keratinocytes currently lists 3038 cellular proteins (2127 isoelectric focusing, IEF; and 911 nonequilibrium pH gradient electrophoresis, NEPHGE) many of which correspond to post‐translational modifications. 763 proteins have been identified (protein, name, organelle components, etc.) and they are listed both in alphabetical order and with increasing SSP number, together with their Mr, pI, cellular localization and credit to the investigator(s) that aided in the identification. Furthermore we have listed 176 proteins that have been microsequenced so far and that are recorded in this database. We also include synthetic images depicting some interesting sets of proteins identified so far; these include components of hnRNP's, proteasomes or prosomes, ribosomes, as well as assorted organelle markers, GTP‐binding proteins, calcium binding proteins, stress proteins, autoantigens, differentiation markers and psoriasis upregulated proteins. The aim of the comprehensive database is to gather, through a systematic study of keratinocytes, qualitative and quantitative information on proteins and their genes that may allow us to identify abnormal patterns of gene expression and ultimately to pinpoint signaling pathways and components affected in various skin diseases, cancer included.
Influence of the electrode compartment separating membranes on continuous flow electrophoresis
Authors: Heinrich Jörn , Wagner Horst 
Published: May 2005

AbstractThe influence of the ion permeable diaphragms, separating the electrode compartments of continuous flow electrophoresis chambers from the separation space, are described in detail. A set of equations is derived from a simplified phenomenological model of the ion transport across media boundaries. These equations are useful to predict the magnitude of concentration changes near the membranes due to the electrophoretic ion transport. Adaption to any CFE geometry, membrane material and electrolyte system is possible with only a few measurements, which yield the transference number difference as a characteristic transport property of the membrane material for a special electrolyte. The validity of the equations is checked by experiments. Local electric field strength and local conductivity are measured with a potential gradient‐conductivity scanner developed in our laboratory. This device yields data on the dynamics of the concentration changes.
Human liver protein map: Update 1993
Authors: Hughes Graham J. , Frutiger Séverine , Paquet Nicole , Pasquali Christian , Sanchez Jean‐Charles , Tissot Jean‐Daniel , Bairoch Amos , Appel Ron D. , Hochstrasser Denis F. 
Published: May 2005

AbstractThis publication updates the reference human liver protein map. By microsequencing, 27 spots or 34 polypeptide chains were identified. The most abundant polypeptides detected on the silver stained liver map were key elements in major hepatic biochemical pathways. The new polypeptides and previously known proteins are listed in a table and/or labeled on the protein map, thus providing the 1993 reference human liver SWISS‐2DPAGE database. SWISS‐2DPAGE and the SWISS‐PROT protein sequence databases are closely linked together through the use of common accession numbers.
How far have we progressed toward automated electrophoresis in sieving media of the twenty‐first century?
Authors: Chrambach Andreas 
Published: May 2005

AbstractThe automation of electrophoresis in polymeric sieving media requires (i) an objective definition of the conditions (the polymer, its concentration, solvent, buffer, pH, ionic strength, temperature) under which a particular separation proceeds most effectively; (ii) apparatus capable of zone detection, acquisition by computer, evaluation (migration distance, zone width and area) and a printout of the number of components, their size and net charge, and the polymer conditions under which each component separates most effectively from its two neighboring zones. Both of these prerequisites of automation have been met to a first approximation at this time and, after further maturation, assembly and streamlining should be able to fill the need of the coming century for a more efficient, nonarbitrary and cost‐effective mode of macromolecular and cellular particle separation. (i) The realization of the qualitative equilivalence of polymer solutions and gels had greatly increased our options in the choice of sieving media. That choice can be made objectively by correlating separation efficiency (S), particle size (R) and intrinsic viscosity (ηo) of the polymer. (S) is a function of the slope, KR (R), of the Ferguson plot [log(mobility) vs. polymer concentration], or with nonlinear plots (DNA, agarose) KR(R,T). KR is at present most easily derived from transverse pore gradient gels or by conducting capillary zone electrophoresis (CZE) at multiple polymer concentrations. Pore gradient CZE appears promising. CZE also defines the free mobility unequivocally. Computer programs exist to generate KR from migration distances (times), and optimal S and polymer concentration for a particular R from KR. Optimal S can be related objectively with ηo to define, as a first‐order approximation, the optimal polymer for the separation within a narrow R‐range. (ii) Apparatus with automated detection of zones and acquisition of migration distances has become commercially available (e.g. Applied Biosystems, Du Pont, LabIntelligence). One of these scans multiple lanes and can be interfaced to compute KR and S. Thus, all elements necessary for automated electrophoretic separation under optimally effective separation conditions are available in rudimentary form. Their development, assembly, integration and streamlining should be feasible prior to the onset of the next century.
Electrophoretic light scattering
Authors: Takagi Toshio 
Published: May 2005

AbstractUnder a microscope, electrophoretic mobilities of visible particles can be determined in a free solution without any boundary or zonal separation. Electrophoretic light scattering based on the Doppler velocimetry can be taken as a nanometer‐scale version of the above traditional electrophoresis. An electrophoretic light scattering apparatus was developed to enable mobility determination of proteins and surfactant micelles which have been outside the range of the electrophoresis.
Electrophoretic artifacts arising from the use of thiol‐containing reagents
Authors: Gianazza Elisabetta , De Ponti Paola 
Published: May 2005

AbstractThiol reagents migrate as a curtain behind the salt front when loaded with the sample solution onto disc‐electrophoresis gels. In immobilized pH gradients (IPG) the same compounds are driven by electrophoresis and electroosmosis from the alkaline to the neutral and acidic regions of the gradients. In either case, a dose‐dependent sideways spreading results in spurious reduction between adjacent lanes if samples with and without reducing agent are loaded side by side.
On the use of dimensionless parameters in acid‐base theory. V. Buffers composed of binary mixtures of monovalent weak acids and bases
Authors: Rilbe Harry 
Published: May 2005

AbstractA general theory for buffers consisting of one weak acid and one weak base is developed, particular interest being devoted to the special case of doubly weak salts. The pH course as it varies with the acid/base concentration ratio is presented in equations and graphs. The influence of ΔpK′ = pK′b−pK′a, which can be negative as well as positive, is discussed and visualized in graphs. The buffer capacity is deduced as a function of pH. The buffer range is, at a maximum, 2.67 times that of a monovalent weak protolyte for ΔpK′ = 2.7 pH units. The buffer capacity curve has two maxima for ΔpK′ values bigger than 0.77. For negative ΔpK′ values the buffer capacity curve has only one maximum, and the buffer range is not better than that of a monovalent protolyte. A general equation for the ionic concentrations is deduced, and as a corollary the degree of hydrolysis of the doubly weak salt is obtained as a function of ΔpK′. For solutions of this salt, the relation between concentration and pH is elucidated by equations and graphs.
Application of an improved density gradient electrophoresis apparatus to the separation of proteins, cells and subcellular organelles
Authors: Tulp Abraham , Verwoerd Desirée , Pieters Jean 
Published: May 2005

AbstractA DGE apparatus, made of Perspex, consisting of a separation column (5 × 2.2 cm) and containing a 0–4% linear Ficoll density gradient, was constructed. Only 2.5 cm of the column were used for high resolution separations. A specially designed removable top cone permitted precise gradient introduction, thin sample layering (0.3–1 mm) and precise fractionation after electrophoresis. A bottom circular palladium anode (nongassing) was separated hydrodynamically but not electrically from the density gradient by a cationpermeable membrane. A top circular platinum cathode caused negatively charged particles to migrate upwards (levitation). Thin sample layering permitted short separation times (30–60 min) at only 3 V/cm (10 mA). As for proteins, glycoforms of α1‐antitrypsin were separated as well as isoenzymes of β‐hexoseaminidase. Furthermore, separation of transferrin (Tf) from the putative Tf‐receptor complex was effectuated. The device was equally suitable for the separation of Megadalton proteins (mucins). Artificial mixtures of intact erythrocytes (rat, rabbit, human) were separated with high resolution. About 107 cells (of 100 μm3 cell volume) could be loaded onto the device. Crude microsomes from the human melanoma cell line Mel JuSo were separated after brief trypsin treatment within 38 min at 10 mA. Ratios of the migration velocities of the constituent organelles were: late endosomes (LE) : lysosomes (L) : Golgi (G) : early endosomes (EE) = 1 : 0.94 : 0.77 : 0.55 and under slightly different conditions LE : L : G : endoplasmatic reticulum (ER) : plasma membrane (PM) = 1 : 0.87 : 0.64 : 0.58 : 0.49.
A fast spot segmentation algorithm for two‐dimensional gel electrophoresis analysis
Authors: Wu Yecheng , Lemkin Peter F. , Upton Kyle 
Published: May 2005

AbstractAn important issue in the automation of two‐dimensional gel electrophoresis image analysis is the detection and quantification of protein spots. A spot segmentation algorithm must detect, define the extent of, and measure the integrated density of spots under a wide variety of actual gel image conditions. Besides these functions, the algorithm must be memory efficient to be able to process very large gel images and do this in a reasonable amount of computation time on low‐cost computers, such as workstations and personal computers. We have developed a fast spot segmentation algorithm, extending the GELLAB‐II segmenter, which extracts spots in a single raster scanning pass through the gel image. The performance analysis of the algorithm will be given in the paper as well as a discussion of the algorithm.
Bronchoalveolar lavage fluid proteins in human lung disease: Analysis by two‐dimensional electrophoresis
Authors: Lenz Anke‐Gabriele , Meyer Barbara , Costabel Ulrich , Maier Konrad 
Published: May 2005

AbstractProteins of human bronchoalveolar lavage fluids, obtained by washing the epithelial lining fluid of the lungs with phosphate‐buffered saline, were analyzed by two‐dimensional electrophoresis under denaturating and reducing conditions. The two‐dimensional pattern of bronchoalveolar lavage fluid proteins of healthy volunteers (controls) were compared with those of patients with idiopathic pulmonary fibrosis, sarcoidosis, and asbestosis. Particular interest was paid to the proteins present in minor amounts mainly in the low molecular weight region of the gels. Marked changes in single protein spots were observed. In idiopathic pulmonary fibrosis the spot intensity of the surfactant‐associated protein, SP‐A, showing isomeric forms both in charge and in molecular weight, was markedly decreased. In sarcoidosis, the immunoglobulins (IgG, IgA) and a group of protein spots at an isoelectric point of 4.5–5.0 and a molecular mass of 55 kDa were increased. An additional spot appeared at an isoelectric point of 4.5 and a molecular mass of 12 kDa. In particular in asbestosis, but also in some cases of idiopathic pulmonary fibrosis and sarcoidosis, the number and intensity of low molecular weight proteins were increased strongly.
Study of pH‐dependent structural transition in telomeric sequences by two‐dimensional gel electrophoresis
Authors: Belotserkovskii Boris P. , Frank‐Kamenetskii Maxim D. 
Published: May 2005

AbstractWe have applied the method of two‐dimensional gel electrophoresis to study a pH‐dependent structural transition in plasmid carrying the Tetrahymena telomeric sequence, (G4T2)n. A study of the two‐dimensional patterns makes it possible to obtain energetic characteristics of the transition. We have obtained the nucleation energy of the structure. It proved to be two times lower than the nucleation energy of H‐DNA determined before for a d(AG)n insert. The finding partly explains the formation of an eclectic structure in telomeric sequences. We also studied the effect of Zn++ ions on the two‐dimensional patterns of gel electrophoresis. Our data show that Zn++ ions can significantly affect the transitions themselves. This finding leads to reinterpretation of numerous data on probing of structural transitions in DNA by the S1 nuclease.
DNA electrophoresis in polymer solutions: Ogston sieving, reptation and constraint release
Authors: Viovy Jean‐Louis , Duke Thomas 
Published: May 2005

AbstractThe electrophoresis of long polyelectrolytes is considered theoretically, with special attention to duplex DNA. We first discuss quantitative approaches to determine unambiguously the entanglement properties of polymer solutions. Following an idea proposed by Grossman and Soane, we then assume that the “mesh” size of the solution plays the role of a dynamic “pore size” in order to apply theories for gel electrophoresis. In the framework of the Ogston model, we predict that duplex DNA up to 1 kb or more should be separable in dilute (i.e. nonentangled) solutions of high molecular weight polymers. In an entangled solution, and for DNA larger than the pore size, we use a recently developed fluctuation‐reptation model to predict the range of sizes in which separation should be possible as a function of electric field E and pore size ξ b. For ξb larger than the Kuhn length of DNA, we predict a separation up to a size N*scaling as E−1ξb−1. For ξb smaller than the Kuhn length, two different regimes are expected. For small electric fields (typically of the order of 10 V/cm), N*should be proportional to E−1ξb−3/3, whereas for high electric fields such as encountered in capillary electrophoresis, we expect that N*is proportional to E−2/5ξb12. These predictions are qualitatively different from earlier ones. Finally, we demonstrate that the finite lifetime of the “pores” in anentangled solution (as opposed to a gel) may lead to a new migration mechanism by constraint release, which is not size‐dependent. In contrast with earlier suggestions, we show that, in general, the concentration should be raised above a minimal value significantly higher than the entanglement threshold c*in order to separate large DNA molecules. We propose expressions for this minimal concentration as a function of DNA and polymer sizes. This model suggests that, for a given high molecular weight polymer, the size of the largest DNA that can be separated increases roughly linearly with the viscosity of the solution.
Cellular automaton simulation of pulsed field gel electrophoresis
Authors: Smith Mark A. , Bar‐Yam Yaneer 
Published: May 2005

AbstractWe describe simulation techniques well suited to detailed investigation of the microscopic behavior of DNA during electrophoretic separation in the diffusive regime. Long polymers moving diffusively in a medium are simulated using microscopic Monte‐Carlo steps. Simulations rely upon a recently introduced two‐space abstract polymer that enables fine‐grained massively parallel simulation. Tests of the two‐space polymer dynamics are reviewed. The scaling with polymer length of the size and relaxation time of isolated polymers are shown to agree with universal scaling relations. The relaxation time is found to be significantly faster than the alternative bond‐fluctuation method. Simplicity of implementation enables simulation on cellular automaton machines (CAM) including CAM‐6, and a prototype of the new CAM‐8, as well as other massively parallel architectures. Preliminary simulations of polymers migrating under an external field through a random medium of obstacles in two dimensions are described. Two sequences of simulations are performed, with different obstacle densities corresponding to pore sizes larger and smaller than the polymer radius of gyration. In the dilute medium polymers are characteristically draped on single obstacles. In the denser medium draping across multiple obstacles results in reduced orientation in the field direction. A demonstration of rapid 90° field direction switching results in polymer motion toward the expected intermediate direction.