bluuebunny/crossref_metadata_2025_split huggingface dataset:
Electrophoresis and densitometry of serum and urine in the investigation and significance of monoclonal immunoglobulins
Authors: Macnamara Elizabeth M. , Whicher John T. 
Published: May 2005

AbstractAbout 15% of monoclonal components or paraproteins are associated with malignancy when detected by simple electrophoresis on cellulose acetate membranes or agarose gel followed by protein staining. More sensitive methods for detecting monoclonal components result in a lower frequency of association with recognisable underlying disease. The sensitivity is dependent on the system used. Isoelectric focusing is 10 to 40 times more sensitive than simple electrophoresis at detecting monoclonal components. Electrophoretically separated bands may be quantitated densitometrically and at the present time this is the most satisfactory method for measuring monoclonal component concentration. Immunochemical methods for quantitating monoclonal components are limited by failure to react in a similar manner to polyclonal standards and giving problems of antigen excess resulting both in falsely elevated and low results. Electrophoretic methods must always be used in conjunction with these. The normal polyclonal ratio of immunoglobulin kappa:lambda light chain is distrubed by the presence of a monoclonal component. In 260 patients with myeloma we found that serum electrophoresis alone detected 90% while a disturbance in the kappa:lambda ratio detected 98%. However, 50 % of monoclonal components less than 3 g/L were missed. Log kappa:lambda ratio correlates well with the densitometric scan of monoclonal components, both in vitro and in patients being monitored for treatment response in myeloma. This approach may complement or even replace densitometry for this purpose in the future.
Two‐dimensional gel electrophoresis, protein electroblotting and microsequencing: A direct link between proteins and genes
Authors: Bauw Guy , Rasmussen Hanne Holm , Bulcke Marc Van Den , Damme Jozef Van , Puype Magda , Gesser Borbala , Celis Julio E. , Vandekerckhove Joël 
Published: May 2005

AbstractWe have used two‐dimensional gel electrophoresis as a general „preparative”︁ method to purify proteins for microsequencing analysis. In the first experiments, proteins derived from a total extract of Nicotiana tabacum leaf tissue were directly blotted from the gel onto poly(4‐vinyl‐N‐methylpyridinium iodide)‐coated glass fiber sheets. The major spots were excised and subjected to NH2‐terminal sequence analysis, which made it possible to identify five of the eight selected proteins, while two more were recognized by generated internal sequences. In a second set of experiments, proteins of human origin were separated on multiple two‐dimensional gels and the Coomassie Brilliant Blue‐stained spots were excised from the gels. The combined spots were re‐eluted and concentrated in a new gel and blotted on Immobilon. They were fragmented by in situ proteolysis and the generated peptides were separated by reverse phase–high performance liquid chromatography and sequenced. At the average, the internal sequences that were obtained covered 35 residues per protein and allowed unambiguous identification of 13 of the 23 proteins analyzed so far. The sequence information obtained of the unidentified proteins is sufficient for further cloning. These results demonstrate that systematic sequence analysis of the major proteins seen in two‐dimensional gels is within the reach of current technologies. This offers a unique opportunity to link information contained in protein databases with known or forthcoming DNA sequence data.
Separ of cationic proteins via charge reversal in capillary electrophoresis
Authors: Wiktorowicz John E. , Colburn Joel C. 
Published: May 2005

AbstractAnalysis of proteins by capillary electrophoresis requires strategies which minimize coulombic interactions with the capillary surface. Thus buffers with pH's above the isoelectric points (pI) of proteins, or near the pI of silanol are required for efficient separation. Covalent modification of the capillary surface is also effective; however, this strategy is technically difficult, abolishes endosmotic flow and suffers from the inherent lability of the siloxane bond. Finally, „dynamic coating”︁ agents, which interact weakly with the capillary surface and therefore, must be included in the separation buffer, suffer from the potential interaction of coating agent with analytes, altering the selectivity of the system. In the following paper, we describe another approach which overcomes all of these difficulties, and demonstrate the ease of use, nondenaturing property, stability and selectivity of the coating strategy with several model protein systems.
Monocyte‐specific esterase isoenzyme demonstrated by isoelectric focusing
Authors: Gignac Suzanne M. , Drexler Hans G. 
Published: May 2005

AbstractUsing horizontal thin‐layer isoelectric focusing in polyacrylamide gels, we separated the isoenzymes of carboxylic esterase (EC 3.1.1.1) of cell extracts prepared from human hematopoietic cells. Isoenzyme bands were visualized by staining with naphthol ester as substrate and coupling to an azo dye. Staining intensities of isoenzymes were quantified by densitometric scanning. On isoelectric focusing in a pH 2–11 gradient, distinct esterase isoenzyme profiles could be discerned and correlated to various types of normal hematopoietic cells and their leukemic counterparts. One unique isoenzyme, termed monoband, could be clearly identified on the basis of its isoelectric point (pI 6.0), its strong expression by normal and malignant monocytes and its complete and selective inhibition by sodium fluoride. This band was only found in monocytes of either normal or leukemic origin, but not in lymphoid or myeloid cells. The monocyte esterase could be inhibited by sodium fluoride whereas other isoenzyme bands were resistant to this inhibition. However, the specificity of this inhibitory reaction was relative, depending on the concentration of sodium fluoride. Compared with normal monocytes, leukemic monocytes often showed an overexpression of the mono‐bands. Dilution experiments established the distinct prominence of the mono‐band which could be detected among the other isoenzymes even when only 1% of the total cell population consisted of monocytes. Immature myeloid, but mono‐band negative leukemic cells whose arrest of differentiation can be overcome by in vitro 12‐O‐tetradecanoylphorbol 13‐acetate‐promoted differentiation to more mature cells, could be induced to express the mono‐band which paralleled their maturation to monocytes. Isoelectric focusing of esterase isoenzymes represents a powerful tool for the isoenzymatic analysis of hematopoietic cells and an enzymological classification of leukemias.
Electrophoretic characterization of posttranslational modifications of human parotid salivary α‐amylase
Authors: Bank Ruud A. , Hettema Ewald H. , Arwert Fré , Amerongen Arie V. Nieuw , Pronk Jan C. 
Published: May 2005

AbstractHuman salivary α‐amylase displays multiple bands upon native polyacrylamide gel electrophoresis. In fresh saliva, due to posttranslational modifications, a pattern of 5–6 isozymes is observed. The isozymes are designated 1–6, in the order of increasing anodal mobilty. As a result of the development of a rapid and sensitive electrophoresis system, with markedly higher resolution than previously reported, we concluded that a previously proposed model (Karn et al., Biochem. Genet. 1973, 10, 341–350) is inadequate to explain the origin of the various bands. We propose an alternative model that fits in with our new and previously made observations. According to this model, band 2 is the primary gene product and band 1 is its glycosylated counterpart with only one neutral oligosaccharide present on each molecule. Band 3 originates from band 1 by the transialidase‐catalyzed incorporation of sialic acid into the biantennary chain. Bands 4 and 6 originate from bands 2 and 4, respectively, by deamidation; band 5 is the deamidation product of amylase with an acidic oligosaccharide (band 3). Only a minor part of band 3 consists of the deamidation product of band 1. Peptide Asn‐Gly‐Ser (residues 427–429) is the most probable candidate for glycosylation; literature data suggests that deamidation occurs in the stretch Glu‐Asn‐Gly‐Lys‐Asp (residues 364–368) and Asn‐Gly‐Asn‐Cys (residues 474–477). Both glycosylation and deamidation might play a role in the clearance of amylase from the systemic circulation. The elecroporesis system described is a powerful tool to determine amylase isozyme distributions in health and disease, especially for the screening of alterations seen in ectopically produced amylase.
Discontinuous buffer system for polyacrylamide and agarose gel electrophoresis of DNA fragments
Authors: Orbán László , Chrambach Andreas 
Published: May 2005

AbstractDNA fragments up to 9 kb in size were stacked and separated by polyacrylamid gel electrophoresis, and those up to 50 kb in size by agarose gel electrophoresis using a discontinuous buffer system. Polyacrylamide gel at pH 8.9, 2°C, 0.01 M ionic strength, yielded sharp bands with DNA loads of 8 μg/cm2 of gel of a mixture of 19 DNA fragments in the size range of 72–23130 bp, while agarose gels at pH 8.5, 25°C, provided well‐resolved, unperturbed bands at 0.04 M ionic strength with DNA loads of 1 μg/cm2 of the same mixture. Note that the ionic strength of the agarose gels is comparable to the conventionally used 0.5 × TBE (Tris‐borate‐EDTA) buffer, while that successfully applied to polyacrylamide is seven‐fold less than the ionic strength of conventionally used 1 × TBE buffer, with is substantially shorter duration of electrophoresis as a result. The application of a discontinuous buffer system to the gel electrophoresis of DNA results in (i) Band identification Rf, the migration distance relative to a sharply defined “buffer front” (moving boundary). This is sufficiently labor saving, compared to determining absolute mobilities, so as to render practical the expression of bands as numbers, with benefits for data storage, statistical manipulations and physico‐chemical exploitation of mobility data. The use of Rf's also circumvents loss of precision in mobility measurement resulting from progressive band spreading of dye bands used as a front. (ii) A uniformly and highly concentrated starting zone, beneficial to resolution, is obtained, without the losses by which separate concentration steps are usually burdened. (iii) The degree of dilution of the DNA sample becomes unimportant.
Des‐gamma‐carboxyprothrombin detection by immunoblotting after polyacrylamide gel affinoelectrophoresis in human plasmas
Authors: Belle Michèle , Hanss Michel , Guillaumont Mark , Leclercq Michel , Guinet Roland 
Published: May 2005

AbstractIn the absence of vitamin K or in the presence of the vitamin K antagonists, abnormal nonfunctional forms of prothrombin circulate in the blood. A reliable and reproducible technique, derived from traditional crossed affinoimmunoelectrophoresis in presence of calcium lactate, was developed and optimized. The technique is based on nondenaturing polyacrylamide gel affinoelectrophoresis, with calcium lactate, of plasma samples, followed by immunoblotting with rabbit anti‐human prothrombin serum and detection with an anti‐rabbit immunoglobulin peroxidase conjugate. Depending on the plasmas, one or two bands were visualized and quantified by densitometry of the immunoblots. The technique was able detect abnormal des‐gamma‐carboxylated prothrombins at concentration of 0.1 μg/mL.
Barley cultivar discrimination: II. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and isoelectric focusing with immobilized pH gradients
Authors: Weiss Walter , Postel Wilhelm , Görg Angelika 
Published: May 2005

AbstractIsoelectric focusing performed with immobilized pH gradients was found superior to other commonly used electrophoretic methods for discrimination of 55 European winter and spring barley cultivars. Hordeins, the alcohol‐soluble proteins, yielded 32 different patterns, allowing identification of 22 cultivars and classification of the remaining ones into ten groups of two to eight cultivars each. Only 21 different hordein patterns were observed using horizontal sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, followed by silver staining. Twelve cultivars exhibited unique hordein patterns, the remaining nine groups contained 2–11 cultivars. Resolution of isoelectric focusing with immobilized pH gradients was further enhanced in some cases when the patterns of urea/dithiothreitol‐soluble proteins were used instead of the hordein patterns. However, evaluation was more complicated because of the larger number of protein bands detected.
Evaluation of membranes used for electroblotting of proteins for direct automated microsequencing
Authors: Baker Cathy S. , Dunn Michael J. , Yacoub Magdi H. 
Published: May 2005

AbstractProtein purification and characterisation have been age‐old problems for the biochemist. A new era has arisen with the advent of one‐ and two‐dimensional gel electrophoresis and high sensitivity automated protein microsequencing. These two tools along with electroblotting have made it possible to separate and analyse complex protein mixtures. We studied six different membranes compatible with Edman degradation chemistry to determine their efficiencies at binding proteins electroblotted from one‐dimensional sodium dodecyl sulphate‐polyacrylamide gel electrophoresis. Their overall blotting‐sequencing properties were also evaluated. We found that the polyvinylidene difluoride‐based membranes out‐performed the glass‐based and polypropylene‐based membranes under our selec ted experimental conditions. The problems associated with electroblotting and microsequencing are discussed.
Identification of human esterase D subunits from the homodimeric and heterodimeric forms of five phenotypes by a new two‐dimensional isoelectric focusing method
Authors: Alonso Antonio , Visedo Guillermo , Sancho Manuel , Fernández‐Piqueras José 
Published: May 2005

AbstractA new two‐dimensional isoelectric focusing method was developed to identify the human esterase D (EsD) subunits from the homodimeric and heterodimeric forms of five EsD phenotypes. EsD polymorphism was also analyzed by one‐diniensional isoelectric focusing under reducing and mild denaturing conditions to study the influence of dithiothreitol and low concentrations of urea on the focusing pattern of the EsD dimers.
On the relationship of amino acid composition to silver staining of proteins in electrophoresis gels: II. Peptide sequence analysis
Authors: Gersten Douglas M. , Rodriguez Lewis V. , George David G. , Johnston Dennis A. , Zapolski Edward J. 
Published: May 2005

AbstractThe quantification of proteins in silver‐stained electrophoresis gels has been limited by the differences in “stainability” of different proteins. Despite efforts by many researchers, the precise basis of the reaction between silver reagents and polypeptides is still unclear, and, depending on the formulation, may even differ. We have tested the hypothesis that differences in stainability among proteins can be attributed to diferences in di‐ or tripeptide composition. The results indicate that some order of protein structure other than short peptides accounts for the staining differences observed.
A technique for detecting antifungal activity of proteins separated by polyacrylamide gel electrophoresis
Authors: De Bolle Miguel F. C. , Goderis Inge J. , Terras Franky R. G. , Cammue Bruno P. A. , Broekaert Willem F. 
Published: May 2005

AbstractA technique was developed for the detection of antifungal activity of proteins after discontinuous polyacrylamide gel electrophoresis under native conditions. The antifungal activity is detected as growth inhibition zones in a homogeneous fungal lawn, grown in an agar layer spread on top of the polyacrylamide gel. The position of proteins with antifungal activity can be determined on a diffusion blot prepared from the same gel. The technique is illustrated for three antifungal plant proteins, i.e. α‐purothionin, Urtica dioica agglutinin, and tobacco chitinase.
Alteration in the electrophoretic mobility of OmpC due to variations in the ammonium persulfate concentration in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis
Authors: Lobos Sergio R. , Mora Guido C. 
Published: May 2005

AbstractStudies on Salmonella typhi and Salmonella typhimurium outer membrane proteins have shown that the relative position of OmpC porin in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis undergoes an important shift when the concentration of ammonium persulfate in the running gel is increased from 6 to 12 mM. The apparent molecular mass at these concentrations was estimated to be 34 and 40 kDa, respectively. Under similar electrophoretic conditions the apparent molecular mass estimated for OmpF was 37.6 an 38.2 kDa. Therefore, OmpC moves from a leading position to a position behind OmpF. For Escherichia coli OmpC the shift observed is less pronounced than that occurring in Salmonellae.
The identification of proliferation and tumour‐induced proteins in human endothelial cells: A possible target for tumour therapy
Authors: Clarke Mark S. F. , West David C. 
Published: May 2005

AbstractThe continued growth and spread of tumours is dependent on the proliferation of the endothelial cells of their vasculature. The presence of proliferation‐ or tumour‐induced surface proteins on these endothelial cells would offer a suitable epitope for monoclonal antibody therapy of tumours. Using cultured human umbilical and capillary endothelial cells, we have stimulated them with simple mitogens and tumour conditioned media and examined the proteins induced by [35S]methionine incorporation and 125I‐surface‐labelling. Two‐dimensional polyacrylamide gel electrophoresis revealed the induction of proliferation and tumour‐related antigens on the surface of the endothelial cells. Subsequent monoclonal antibody studies suggest that tumour specific surface proteins are present on most tumour endothelium.
Quantitation of human leukocyte proteins after silver staining: A study with two‐dimensional electrophoresis
Authors: Giometti Carol S. , Gemmell M. Anne , Tollaksen Sandra L. , Taylor John 
Published: May 2005

AbstractThe quantitative attributes of human leukocyte proteins detected by silver staining two‐dimensional electrophoresis (2‐DE) gels were studied by using computerassisted data analysis. Experiments included (a) analysis of replicate patterns of the same sample, (b) analysis of different dilutions of the same sample, and (c) analysis of samples from different individuals. Over 200 proteins were observed to have coefficients of variation (CV) less than or equal to 15 % when data from replicate patterns were analyzed. In contrast, 8 proteins had CV values of less than or equal to 15 % when data from different samples were analyzed. The dilution experiment showed that a majority of the proteins detected with some consistency (i.e., observed in at least 80 % of the patterns) have a linear relationship between the amount of protein loaded onto a 2‐DE gel and the spot volume in the final 2‐DE pattern. The slope of the curves and the deviation from linearity were found to be quite protein‐specific. These results indicate that optimization of sample purity and minimization of staining protocol variables are required to limit the background quantitative variability between and within 2‐DE runs to a level that will allow detection of quantitative changes indicative of biological responses.
Computer‐aided calculation of the molecular size of nondenatured proteins in pore‐gradient gel electrophoresis
Authors: Rothe Gunter M. , Weidmann Hans 
Published: May 2005

AbstractA computer program written in Turbo C is described, which uses the two‐step mathematical procedure published recently (Rothe, G. M., Electrophoresis 1988, 9, 307–316) to evaluate the molecular mass, Stokes'radius, spherical radius, and frictional coefficient of nondenatured proteins. The program runs on any IBM‐PC or 100% compatible IBM‐PC, provided the disk operating system MS‐DOS or PC‐DOS 3.0 or later has been installed. Functions that are permanently in use are accessible by menu. Storage and loading of data from disk and help instructions can be called by use of function keys. The program provides several tables into which inserted and calculated data is automatically integrated. Each table can be printed out, provided a printer with IBM character set is connected to the computer.
The master two‐dimensional gel database of human AMA cell proteins: Towards linking protein and genome sequence and mapping information (Update 1991)
Authors: Celis Julio E. , Leffers Henrik , Rasmussen Hanne Holm , Madsen Peder , Honoré Bent , Gesser Borbala , Dejgaard Kurt , Olsen Eydfinnur , Ratz Gitte P. , Lauridsen Jette B. , Basse Bodil , Andersen Annette H. , Walbum Else , Brandstrup Birgitte , Celis Ariana , Puype Magda , Van Damme Josef , Vandekerckhove Joël 
Published: May 2005

AbstractThe master two‐dimensional gel database of human AMA cells currently lists 3801 cellular and secreted proteins, of which 371 cellular polypeptides (306 IEF; 65 NEPHGE) were added to the master images during the last 10 months. These include: (i) very basic and acidic proteins that do not focus under normal running conditions and (ii) low‐abundant proteins that can only be detected after prolonged gel exposure. Annotation categories updated in this version include “protein name”, “antibody against protein”, “cellular localization”, and “microsequenced proteins”. New entries include “human autoantigens” and “cDNAs”. For convenience we have included an alphabetical list of all known proteins recorded in this database. In the long run, the main goal of this database is to link protein and DNA sequencing and mapping information (Human Genome Program) and to provide an integrated picture of the expression levels and properties of the thousands of proteins that orchestrate various cellular functions both under physiological and abnormal conditions.
High resolution‐separation of DNA bands by electrophoresis with a long gel in a fluorescence‐detection DNA sequencer
Authors: Nishikawa Tetsuo , Kambara Hideki 
Published: May 2005

AbstractA high‐resolution separation of DNA bands is achieved by electrophoresis with a long gel in DNA base sequencing using fluorescence detection. We separate 760 and 761 base DNA fragments using the 93 cm migration electrophoresis optimized for the separation of DNA bands. A T7 DNA polymerase and an Mn++ buffer are used in sequencing reactions to obtain fluorescence peaks of uniform strength, and the peak areas in the spectrum are used for recognizing the peak number in a cluster of successive peaks. This method is successfully applied to the DNA fragment spectrum obtained by 93 cm migration electrophoresis, which results in a single‐band differentiation of bands of 1040 base DNA.