bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of intracellular deoxyribonucleases using polynucleotide‐polyacrylamide gel electrophoresis
Authors: Brown Glenn E. , Karpetsky Timothy P. , Rahman Aneesa , McFarland Edward , Haroth Mary B. 
Published: May 2005

AbstractIntracellular deoxyribonucleases (DNases) in human peripheral blood leukocytes have been identified and characterized by polynucleotide‐polyacrylamide gel electrophoresis (PPAGE). This technique combines the protein separating powers of polyacrylamide gel electrophoresis with the sensitivity of detection characteristic of an enzyme assay. Visualization of nuclease activity is made possible by the inclusion of high molecular weight DNA substrate within the sieving portion of a narrow polyacrylamide gel. No sodium dodecyl sulfate, urea, or other protein denaturants are used in this procedure. However, electrophoresis of the contents of up to 106 cells/gel is accomplished using conditions under which the enzymes are inactive. The positions of DNases are then revealed by incubation of the gel in an appropriate buffer followed by staining for intact DNA. Colorless regions correspond to the presence of enzyme. Used as a pattern recognition tool, PPAGE revealed that leukocytes from normal donors contained lower levels of certain activities than did cells from donors with chronic myelogenous leukemia. The rapid simultaneous characterization of multiple activities is made possible by simply using different buffer compositions in the incubation step. For example, ionic strength and pH optima may be found directly for activities present in unpurified human intracellular contents. The reproducibility of this method permits comparison of individual activities by using Ferguson plot analysis to detect differences or similarities among enzymes. These analyses revealed that DNase activities in human peripheral blood leukocytes fell into a minimum of two families. Modifications of overall effective charge probably account for microheterogeneity.
Photometric evaluation of slab gels II: Delimitation and integration of peaks in one‐dimensional electropherograms
Authors: Yakin Habib M. , Kronberg Harald , Zimmer Hans‐G. , Neuhoff Volker 
Published: May 2005

AbstractIn order to increase accuracy and precision of quantifying one‐dimensional electropherograms by digital signal processing, a set of algorithms is presented that operate on the optical density values acquired by a linear scanning densitometer. These operations include smoothing of the measuring values, background correction by an optimized piecewise linear baseline, and integration of the peak areas after two‐sided fit of half‐Gaussian profiles to each stained protein band. The algorithms are implemented as BASIC‐programs and are readily adaptable to a variety of electrophoretic separations. The procedure has been applied successfully in research [1] as well as in clinical evaluations [2].
Crude‐tissue‐extract analysis by micro‐electrophoresis in cylindrical continuous polyacrylamide gradient gels
Authors: Murach Karl‐Friedrich , Beltle Wolfgang , Bopp Martin 
Published: May 2005

AbstractContinuous gradient gels in capillaries with acrylamide concentrations ranging from 2–20 % or alternatively from 2–40 % are equally suited for the fractionation of complex mixtures of macromolecules in aqueous solutions within a range of molecular weights from 103‐106. A time‐saving method for the simultaneous preparation of up to 48 identical linear micro‐gradient polyacrylamide gels is described. With this method it was possible to compare the patterns of aqueous extracts and to determine tissue‐specific bands of the mossFunaria hygrometrica L. Sibth.in the low molecular weight range for the first time.
Formation of natural pH gradients in sequential moving boundary systems with solvent counterions II. Predicted and experimental properties
Authors: Buzás Zsuzsanna , Hjelmeland Leonard M. , Chrambach Andreas 
Published: May 2005

AbstractSequential moving boundary electrophoresis with protons and hydroxyl ions as the sole counterions, consisting solely of six acids (system A) and of six bases (system B), gives rise to natural steady‐state pH gradients similar to those predicted by theory (pH 2.4–5.5 for system A, pH 10.3–12.2 for system B). Mixtures of the constituents in system A and system B gave rise to a natural pH gradient spanning the pH range between the terminal electrolytes (pH 2.4 and 12.2) predicted for systems A and B. The predicted boundary displacement is negligibly small. Experimentally the boundary displacement could not be determined in view of the difficulty of separating it from the initial transient state in which the pH gradient is formed, and a final state in which the pH gradient decays.
Anomalous conductivity zones in electrophoresis I. Basic theory for two‐ion systems
Authors: Spencer Michael 
Published: May 2005

AbstractThe progressive increase in circuit resistance observed at times in polyacrylamide gel electrophoresis is attributable in part to the formation of zones of altered salt concentration at the interfaces between gel and solution. Similar changes have been reported for electrophoresis through sucrose gradients. Such zones must develop wherever migrant ions suffer a change in transport number. The paper develops a basic theory for such concentration changes. It is shown that a moving boundary will tend to arise at any interface between two media, leaving behind it a zone of altered concentration. The effect will in general be most noticeable at low ionic strengths and with electrolytes whose ions differ considerably in size, a condition applying to many common buffers. Where both ions are relatively small, diffusion is likely to obscure the boundary movement, but this will not affect the integrated change in concentration. The effect could have various deleterious consequences in gel electrophoresis, particularly in preparative apparatus. There are also implications for techniques such as isoelectric focusing and for the analysis of membrane phenomena.
Anomalous conductivity zones in electrophoresis. II. Theory for three‐ion systems and for changes in pH
Authors: Spencer Michael 
Published: May 2005

AbstractThe theory developed in the previous paper is extended to cover a system containing three ions. If an “inactive” electrolyte whose ions are equally affected by a retarding medium is added to a “zone‐former” for which this is not the case, the main effect is to reduce the integrated concentration changes at the interfaces. Where a buffer contains two ions carrying charges of the same sign, the theory predicts a progressive change of pH within a zone of altered concentration. Other sources of pH change are also considered. Where a buffer includes an uncharged weak acid or base, changes in ionic concentration at an interface will lead to changes in pH. Other changes may arise from the migration of water ions, whose contributions are normally neglected. This is not justified where there are large discontinuities in conductivity. Expressions are derived for the changes in pH under different circumstances. In some cases no steady‐state is possible and there are accelerating shifts of pH at the boundaries of a zone. This could have important consequences in practical electrophoresis.
Detection of subnanogram quantities of DNA and RNA on native and denaturing polyacrylamide and agarose gels by silver staining
Authors: Guillemette Joseph G. , Lewis Peter N. 
Published: May 2005

AbstractA silver staining method is described for detecting subnanogram amounts of DNA and RNA on polyacrylamide and agarose gels. The key step for increased sensitivity and linearity is an initial soaking of the gel in a 0.1% solution of cetyltrimethylammonium bromide (CTAB). Agarose‐containing gels can be silver stained after treatment with formaldehyde. The methods described are comparatively rapid, extremely sensitive (≅ 150 pg of DNA) and inexpensive (0.4 g of AgNo per 25 ml of slab volume).
Two‐dimensional electrophoresis in the development of clinical laboratory tests
Authors: Young Donald S. , Tracy Russell P. 
Published: May 2005

AbstractBefore any new test should be introduced into clinical laboratory practice the need for the test should be carefully assessed. Generally, new tests should only be made available when they have distinct advantages over other tests to assess the same organ function or disease process. The appropriateness of the test must be considered in relation to its intended application and it must be examined in the proper clinical context when it is advocated as a diagnostic test. The potential applications of two‐dimensional electrophoresis in the clinical laboratory have been examined. These include its actual use as a diagnostic test with serum and urine specimens, if the simultaneous examination of several proteins can be justified, and if an important role in the identification of disease related changes in protein concentrations can be established which could subsequently be exploited to develop specific diagnostic tests; then the method has a valid application in the search for new clinical procedures.
Isolation by free‐flow electrophoresis and immunological detection of troponin T from turkey muscle: An application in food chemistry
Authors: Schweiger Anton , Baudner Siegfried , Günther Herbert O. 
Published: May 2005

AbstractThe identification of turkey meat in food products has been accomplished using specific antisera against turkey troponin T. This protein (Mr 37 000) was extracted from fresh muscle tissue and isolated by a procedure that included free‐flow electrophoresis as final fractionation step. Preparations of troponin T with purities of more than 95 % were applied as antigen in the immunization of rabbits. Several standard immunological techniques were used to determine additions of turkey meat in food products heated to different temperatures (including roasting temperature). That way, it was possible to also detect additions of chicken meat on the basis of a partial identity between troponins T from turkey and chicken muscle.
Nonspecific binding of p‐phenylenediamine‐pyrocatechol peroxidase substrate to electrofocused proteins
Authors: Cantarow Walter D. , Saravis Calvin A. , Sampson Christian E. 
Published: May 2005

AbstractNonspecific staining of electrofocused protein bands was found with the peroxidase product of Hanker‐Yates reagent (a mixture of p‐phenylenediamine and pyrocatechol). No difficulty was encountered with diaminobenzidine as peroxidase substrate.
Pseudo‐ligand affinity chromatography and high‐voltage isoelectric focusing as a multiparameter method for separation and identification of plasma proteins
Authors: Allen Robert C. , Arnaud Philippe 
Published: May 2005

AbstractA system is described that utilizes physical‐chemical characteristics of macromolecules, not restricted to charge‐size variations, for the separation of the plasma proteins. The first step, using pseudo‐ligand affinity chromatography, separates the plasma proteins into fractions which were characterized by fused rocket immuno‐electrophoresis. Then electrophoresis in the form of high voltage gradient isoelectric focusing on ultrathin‐layer polyacrylamide gels is employed to further separate each of the fractions by charge differences. The advantages of this technique are that the proteins are maintained without noticeable denaturation, allowing a variety of direct functional tests. One is not restricted to only charge‐size variations for separation and the technique is a preparative‐to‐analytical procedure. Furthermore, the higher voltage gradients employed increase resolution of proteins significantly and require separation times of only 30–35 min. The limit of detectability is 50 μg/dl of protein, using silver diamine staining. The feasibility of this and related techniques as applied to the study of genetic polymorphisms and as an aid in the correlation and identification of spots obtained in dissociating‐denaturing two‐dimensional electrophoresis methods is emphasized.
Two‐dimensional polyacrylamide gel electrophoresis of nuclear proteins in human meningiomas
Authors: Unteregger Gerhard , Zang Klaus D. , Issinger Olaf‐Georg 
Published: May 2005

AbstractThe nuclear protein fraction from human meningioma cells was compared with the nuclear protein fraction of fibroblasts, derived from the same patients. The protein pattern obtained was visualized either with the silver stain technique or from labelled cells by fluorography. Whereas no specific polypeptides corresponding to tumor cells or fibroblasts could be detected in one‐dimensional polyacrylamide gel electrophoresis (1‐DE), small differences occur in the two‐dimensional gel patterns (2‐DE), using the silver protein detection. Cell cultures which were labelled during phase G 1 show twice as many polypeptides on the 2‐DE electropherogram with respect to the silver‐stained gels. Besides several proteins detectable in tumor cells and fibroblasts with both methods, a set of 5 polypeptides with an apparent molecular weight (Mr) of 30 000 and an isoelectric point (pI) near 6.1 could be detected in the 2‐DE pattern from labelled tumor cells. Furthermore, the tumor cells showed a remarkably enhanced incorporation of label in proteins in the pH range 5.5–7.0 and with Mr from 14 000–40 000. The fluorograms from both cell types reveal more than 40 polypeptides with a pI of 4.5–5.5 and an Mr from 12 000–45 000, which had never been detected on the silver‐stained electropherograms. The results indicate that cells derived from meningiomas exhibit a pattern of nuclear proteins quite similar to that obtained from fibroblasts with the same diploid karyotype. Furthermore, it seems necessary to combine the high‐resolution separation techniques with other analytical methods, e. g. specific labelling procedures, to enlighten the role of nuclear proteins during the onset of tumorgenesis.
Avidin biotin amplified immunoperoxidase staining of angiotensin‐1‐converting enzyme transferred to nitrocellulose after agarose isoelectric focusing
Authors: Lanzillo Joseph J. , Stevens Joanne , Tumas John , Fanburg Barry L. 
Published: May 2005

AbstractAngiotensin‐1‐converting enzyme (ACE) subjected to isoelectric focusing in agarose gel can be transferred to nitrocellulose in a reliable and reproducible manner. Specific detection of ACE is enhanced 2‐fold by replacing enzyme‐conjugated reagents with avidin biotin‐conjugated reagents. Sensitivity is not diminished when the carcinogenic substrate 3,3′‐diaminobenzidine is replaced with the non‐carcinogenic substrate 3‐amino‐9‐ethylcarbazole. This improved procedure was used to monitor the spontaneous change in the isoelectric point of ACE which occurred during purification of ACE from outdated human plasma.
An improved and simplified apparatus for protein extraction and concentration from gel slices, using moving boundary electrophoresis
Authors: Lan Birgit An Der , Horuk Richard , Sullivan James V. , Chrambach Andreas 
Published: May 2005

AbstractA previous design of an apparatus for extraction and concentration of proteins from gel slices by moving boundary electrophoresis has been simplified, making it into an upper buffer reservoir accessory for the multifunctional gel tube apparatus commonly used for gel electrophoresis. The design of the collection cup has been improved to facilitate assembly and thereby reduce the risk of leakage. The performance of the apparatus was evaluated by the isolation from sodium dodecyl sulfate ‐ polyacrylamide gel electrophoresis of radiolabeled bovine serum albumin and glucagon receptor.
High resolution electrophoresis of extracts of a baby hamster kidney cell line before and after transformation by polyoma virus
Authors: Marshall Thomas , Latner Albert L. 
Published: May 2005

AbstractThe polypeptide patterns obtained following denaturation and high resolution two‐dimensional electrophoresis of proteins from baby hamster kidney cells before and after transformation by Polyoma virus have been compared by silver staining incorporating methylamine. The polypeptides were resolved either by non‐equilibrium pH‐gradient electrophoresis or isoelectric focusing in the first dimension. Two different approaches to isoelectric focusing were employed in order to achieve satisfactory resolution over a wide range of isoelectric point. Both cell lines gave remarkably similar two‐dimensional patterns of spot distribution which allowed changes to be detected in the relative abundance of a number of polypeptides. None of the polypeptides detected were specific to either of the two cell lines.
Effects of chemical exposure on rat serum proteins revealed by a modified technique of two‐dimensional gel electrophoresis
Authors: Marshall Thomas , Vesterberg Olof 
Published: May 2005

AbstractRat sera were analysed by high resolution two‐dimensional electrophoresis to detect polypeptides sensitive to chemical exposure. Exposure to carbon tetrachloride, trichloroethylene or dimethylformamide increased the intensity of a polypeptide with electrophoretic characteristics (pI 4.3, isoelectric point; molecular weight, MW 45 000) similar to those of human orosomucoid. The two‐dimensional procedure was modified by using only selected gel lengths (pH range 4.5–6.5) from the first‐dimension isoelectric focusing gels. Three such gel sections were positioned side by side on the top of a second‐dimension gel slab prior to electrophoresis. Using this approach, heavily stained regions of the two‐dimensional pattern were clarified by allowing simultaneous analysis in one gel slab of the same sample at three different dilutions. With this modification other potential marker proteins for chemical exposure were detected. The advantages and application of this modified technique are discussed.
Enzymatic amplification of electroimmunoprecipitates in agarose gels with horseradish peroxidase‐labeled protein A
Authors: Taketa Kazuhisa 
Published: May 2005

AbstractElectroimmunoprecipitates in agarose gels were amplified with protein A‐horseradish peroxidase (HRP) conjugate. The method is simple and detected quantitatively as low as 0.5 ng of human α‐fetoprotein (AFP) in electroimmunodiffusion. The alternative methods employing the second antibody, labeled initially or afterwards with HRP, failed to stain AFP immunoprecipitates. Direct application of anti‐AFP immunoglobulins‐HRP in electroimmunodiffusion produced less contrasted colored precipitates. Immunoprecipitates of other proteins were similarly visualized by the protein A‐HRP method, avoiding the use of specifically labeled antibody.
Distribution of plasminogen allotypes in eight populations of the Western Hemisphere
Authors: Dykes Dale , Nelson Mary , Polesky Herbert 
Published: May 2005

AbstractPhenotyping for plasminogen (PLG) variants on 0.5 mm agarose isoelectric focusing gels enabled us to identify six different allotypes among the eight populations studied. PLG *1 and PLG *2 were seen at polymorphic frequencies in all the populations studied except Eskimos which had a gene frequency of 0.993 for PLG *1. The variants PLG *3, PLG *B, were seen at low frequencies in some of the populations, whereas PLG *M was only identified in the local White population. A new variant named PLG *D, which we consider to be a marker of black populations, was identified in local Blacks and Black Caribs at frequencies of 0.037 and 0.058, respectively.
High resolution comparison of Plasmodium knowlesi clones of different variant antigen phenotypes by two‐dimensional gel electrophoresis and computer analysis
Authors: Howard Russel J. , Aley Stephen B. , Lemkin Peter F. 
Published: May 2005

AbstractAntigenic variation of the schizont‐infected cell agglutination‐antigen on erythrocytes infected by Plasmodium knowlesi is due to the expression of different malarial proteins. Here we examined whether antigenic variation is accompanied by multiple phenotypic changes in the proteins synthesized by P. knowlesi. The malarial proteins of two clones, one of which was produced by antigenic variation from the other clone in vivo, were labeled by [3H]isoleucine uptake during in vitro parasite growth. The proteins were separated by two‐dimensional gel electrophoresis, detected by fluorography and compared using the GELLAB computer system. In the molecular weight range 35 000 to 230 000 and pH range 4.55 to 6.10, we found only four qualitative differences and two robust quantitative differences among approximately 500 proteins characterizing the protein phenotype of these clones. One clone exhibited four minor protein spots absent from the other. We conclude that antigenic variation in P. knowlesi represents the differential expression of a very small number of malarial genes.
A simple method for drying polyacrylamide slab gels using glycerol and gelatin
Authors: Popescu Octavian 
Published: May 2005

AbstractA method for drying polyacrylamide slab gels of various size and gel composition (T 7.5–15 %, C 2.67 %) is described. The destained gels, containing glycerol, are overlayered with gelatin and dried. Transparent and flexible sheets are obtained which can be stored for extended periods and may also be used for autoradiography.
Detection of endoproteinases in polyacrylamide gels
Authors: Petersen Gary R. , Van Etten James L. 
Published: May 2005

AbstractA simple and versatile method was developed for detecting endoproteinases by nondenaturing polyacrylamide gel electrophoresis. A vertical strip of the resolving gel is subjected horizontally to a second electrophoresis in the presence of a substrate protein and sodium dodecyl sulfate (SDS). The location of proteolytic activity in the original gel strip is revealed by the disappearance of substrate and the appearance of proteolytic products (due to limited proteolysis) in the second gel. The pattern of peptide fragments produced permits the identification of the protease responsible.
Molecular size and charge characterization by rheophoresis I. Theory and gel calibration
Authors: Waldmann‐Meyer Henrik , Pedersen Per Amstrup 
Published: May 2005

AbstractRheophoresis, i.e. thin‐layer electrophoresis in particulate gels with controlled evaporation, combines in one experiment the chromatographic and the electrophoretic characteristics of macromlecules in such a way that they can be determined independently of each other. In this paper theory and experimental corroboration for molecular size determination are presented. We describe the migration velocity of any molecule as the sum of an electrophoretic, an electroosmotic and a rheophoretic component. The latter is the velocity originating from the liquid flow produced by evaporation. It is a function of the position along the gel axis and the cross‐section available to the molecule, but totally independent of the molecular charge. As a result of the position dependence, the rheophoretic component can be isolated from the two others and the available cross‐section determined by means of a simple plot. Thus, the chromatographic partition coefficient is readily calculated and the effective hydrodynamic radius of the molecule obtained.