bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of p53 point mutations by single strand conformation polymorphism: Analysis by capillary electrophoresis
Authors: Atha Donald H. , Wenz H.‐Michael , Morehead Harvard , Tian Jing , O'Connell Catherine D.
Published: May 2005

We have analyzed five p53 single point mutations by single strand conformation polymorphism using capillary electrohoresis (CE‐SSCP) and have compared these measurements to measurements obtained by slab gel electrophoresis (SG‐SSCP). PCR primers were used for amplification of specific exons for mutation detection. 5′ Primers were labeled with FAM (5‐carboxyfluorescein) and 3′ primers were labeled with JOE (2′,7′‐dimethoxy‐4′,5′‐dichloro‐6‐carboxyfluorescein). CE‐SSCP was performed using the Perkin Elmer ABI PRISM™ 310 Genetic Analyzer with GeneScan™ Software and the Beckman P/ACE™ 5510 CE equipped for laser‐induced fluorescence detection. Although the shifts in migration times for the p53 mutations relative to the corresponding wild‐type strands could be successfully detected by either SG or CE analysis, the individual electrophoresis run times were about tenfold faster and more automated with capillary electrophoresis. The CE‐SSCP measurements were performed at temperatures ranging from 10 to 60°C on a prototype instrument. For mutations measured at ambient temperature (25°C), characteristic shift in direction and magnitude were observed in the migration times to both strands of all mutations relative to the wild type. This demonstrated the ability of CE at ambient temperature to resolve these mutations. However, the magnitute and direction of shifts in migration time varied with temperature in a discrete pattern for each mutation and resulted in a temperature‐specific profile for each mutation. This demonstrated that extended temperature control will be an important advantage in resolving single point mutations by CE‐SSCP. In addition, by using CE, discrete intra‐strand isoforms could be easily observed at different temperatures. The combination of mutation‐specific temperature profiling and analysis of isoforms by CE‐SSCP should be of help to the diagnostic community in the detection of genetic mutations.
Enhanced detection sensitivity of “fluorescence reduction” by shifting the analyte absorbance spectrum and use of a fluorescent paper with higher signal/noise ratio
Authors: Yarmola Elena , Chen Nong , Yi David , Chrambach Andreas
Published: May 2005

Nonfluorescing protein bands can be detected by the fluorescence optics of the commerical gel electrophoresis apparatus with automated scanning of the migration path (HPGE‐1000, LabIntelligence, Belmont CA), taking advantage of the decrease of emission from a fluorescent paper placed below the gel by the absorbance of proteins (“fluorescence reduction”). That decrease of fluorescence gives rise to an inverted protein peak. Nonfluorescent colorless proteins appear to reduce the intensity of light emitted from the fluorescent paper due to absorbance of incident and emitted light. When the absorbance spectrum only slightly overlaps with the excitation and emission spectra of the fluorescent paper, that reduction is weak, and detection sensitivity in that application is consequently only 1/30 of that of fluorescent proteins. By contrast, when the protein is colored so that its absorbance spectrum overlaps widely with the excitation and emission spectra of the fluorescent paper, the sensitivity of “fluorescence reduction” equals 1/4 to 1/5 of that obtained for fluorescent proteins. Bands detected by “fluorescence reduction” provide a quantitative measure of protein load and mobility. The area of the inverted bands is proportional to protein loads up to 16 μg/lane of the gel tray. A theory of “fluorescence reduction” is presented which accounts for the existence of a linear relationship between band area and load.
Viscosity‐adjustable block copolymer for DNA separation by capillary electrophoresis
Authors: Wu Chunhung , Liu Tianbo , Chu Benjamin
Published: May 2005

The viscosity‐adjustable property of F127 block copolymer PEO99PPO69PEO99, PEO and PPO being poly (ethylene oxide) and poly(propylene oxide), respectively, was found to be useful for the development of automated capillary electrophoresis (CE). The polymer solution can form a gel‐like structure with sieving ability and can also serve as a dynamic coating material, thereby effectively suppressing the electroosmotic flow induced by the ionization of the silanol group on the quartz capillary inner wall. When applied to CE as a separation medium, F127 block copolymer can provide the advantages of high separation resolution, easy injection and replacement of the triblock copolymer solution and convenient capillary column treatment. High reproducibility of DNA electrophoretic migration time in CE by replenishing F127 solution in acid‐washed capillary tubings can be achieved. The relative standard deviation of the DNA migration time is less than 2%. In the investigation of F127 concentration and temperature effects on the performance of DNA separation in CE, we have found that the DNA electrophoretic migration behavior in the F127 gel‐like solution cannot be described by any one of the existing models.
Characterization of high molecular mass linear polyacrylamide powder prepared by emulsion polymerization as a replaceable polymer matrix for DNA sequencing by capillary electrophoresis
Authors: Goetzinger Wolfgang , Kotler Lev , Carrilho Emanuel , Ruiz‐Martinez Marie C. , Salas‐Solano Oscar , Karger Barry L.
Published: May 2005

In a previous paper, a 2% w/w replaceable high molecular mass linear polyacrylamide solution (high molecular mass LPA) was used to achieve long readlengths for DNA sequencing by capillary electrophoresis (E. Carrilho et al. Anal. Chem. 1996, 68, 3305–3313). In that work, the polymer was prepared by polymerization in water at 6% w/w, followed by dilution to 2% w/w. In this study, an improved method for preparation of high molecular mass LPA was developed, based on inverse emulsion polymerization. With this polymerization procedure, the LPA results in a molecular mass of approximately 9 MDa with characteristics of a fine powder of high purity and practically unlimited shelf life. Using size exclusion chromatography (SEC) and viscosity measure ments to characterize the polymer, good batch‐to‐batch reproducibility was found. It was observed that the viscous polymer solutions made from these high molecular mass polymers require careful preparation and handling because the method of dissolution could affect the molecular mass distribution and the resultant separation of DNA components. Solution containing 2% w/w of LPA made by emulsion polymerization were simple to prepare resulting in excellent performance as a replaceable matrix for DNA sequencing by capillary electrophoresis. The viscosity of the polymer decreased exponentially when pressure was applied. allowing easy replacement from a capillary using a syringe. With a properly prepared matrix, a read‐length on morethan 1000 bases in 80 min with an accuracy better than 97%, and better than 99% for the first 800 bases, could be achieved.
Selenium speciation by interfacing capillary electrophoresis with inductively coupled plasma‐mass spectrometry
Authors: Michalke Bernhard , Schramel Peter
Published: May 2005

The high resolution potential of capillary electrophoresis (CE) makes CE techniques valuable for separations of selenium species. Further, inductively coupled plasma‐mass spectrometry (ICP‐MS) affords element‐specific multi‐element detection, providing extremely low detection limits. The combination of CE with ICP‐MS promises to become a powerful tool for metal speciation. Therefore, an on‐line hyphenation of CE with ICP‐MS, which was developed earlier (Michalke, B., Schramel, P., Fresenius' J. Anal. Chem. 1997, 257, 594‐599), was modified and applied to selenium speciation. For this reason, capillary zone electrophoresis (CZE) methods were developed, providing the possibility to analyze six Se species of interest in one run: Se (IV), Se (VI), selenium carrying glutathione (GSSeSG), selenomethionine (SeM), selenocystine (SeC), and selenocystamine (SeCM). The final CE method used an alkaline background electrolyte (Na2CO3/NaOH) with separation differentiated from the detection step during hyphenation. This resulted in short separation times (10 min) and a subsequent detection step of 100s. The Se species were sufficiently separated from each other and appeared at 7s (SeCM), 16s (Se (VI)), 22s (SeC), 27s (Se (IV)), 35s (SeM) and 56s (GSSeSG) during the detection step. Detection limits were calculated as 10 or 20 μg Se/L for inorganic Se species and 35–50 μg Se/L for organic Se species.
Quantitative trace analysis of L‐ascorbic acid in human body fluids by on‐line combination of capillary isotachophoresis and zone electrophoresis
Authors: Procházková Andrea , Křivánková Ludmila , Boček Petr
Published: May 2005

On‐line combination of capillary isotachophoresis and zone electrophoresis performed in two coupled capillaries (ITP‐CZE) is used for the trace analysis of L‐ascoribic acid in human serum, urine and stomach fluid. At the ITP stage, anionic sample components are separated into individual zones and macrocomponents are detected and driven out of the migration path. In the CZE stage, only a small segment of the sample zones containing L‐ascorbic acid is analyzed. High sensitivity of this hyphenated method (limit of detection, 0.09–0.15 mg/L), low sample volume consumption (2 μL), and acceptable reproducibility of the results (relative standard deviation, 8%) in the concentration range 0.1–15 mg/L demonstrate that the method is applicable for the study of the relation between the content of L‐ascorbic acid in body fluids and the state of health of a person, in which lower amounts of L‐ascorbic acid than the normal levels (i.e., 501–15.1 mg/L in human serum and 12.5–26.8 mg/L in urine) are expected. Possible interferences of other components of the body fluids are excluded by good correlation of the results obtained by the ITP‐CZE method and a routine colorimetric method.
Analysis of changes in acute‐phase plasma proteins in an acute inflammatory response and in rheumatoid arthritis using two‐dimensional gel electrophoresis
Authors: Doherty Niall S. , Littman Bruce H. , Reilly Karen , Swindell Archie C. , Buss Jane M. , Anderson N. Leigh
Published: May 2005

Two‐dimensional (2‐D) gel analysis was used to examine differences in the levels of 19 plasma proteins: before and after an acute inflammatory reaction (parenteral typhoid vaccination) in normal subjects, between rheumatoid arthritis (RA) patients and normals and in RA patients treated with tenidap (120 mg) and piroxicam (20 mg). Typhoid vaccination increased levels of SAA, haptoglobin α1, haptoglobin α2, haptoglobin β and α1‐anti‐chymotrypsin but decreased transthyretin and apolipoprotein E. In RA patients, serum amyloid A (SAA), haptoglobin α2, haptoglobin β, α1‐antichymotrypsin and C3 proactivator levels were elevated while apolipoprotein A‐I, apolipoprotein A‐IV, transthyretin, Gc‐globulin, α2‐HS glycoprotein, α2‐macroglobulin and α1‐B glycoprotein levels were decreased, compared to normals. Compared to piroxicam, tenidap lowered levels of α1‐antiprotease and SAA but raised the levels of transthyretin, Gc‐globulin, α2‐HS‐glycoprotein and α2‐macroglobulin in RA patients. C‐reactive protein (CRP) could not be quantified on 2‐D gels but, When measured by rate nephelometry, levels were reduced after treatment with tenidap compared to piroxicam. The general pattern of the acute phase protein response to an acute inflammatory response to typhoid vaccination is similar to that in the chronic inflammatory condition, RA. The impact of tenidap on both positive and negative acute‐phase proteins in RA patients could clearly be distinguished from that of piroxicam.
Dynamic complexation of solutes in capillary electrophoresis
Authors: Bowser Michael T. , Chen David D. Y.
Published: May 2005

The analyte migration behavior in any chemical separation system can be described using a single equation that unifies all areas of separation science. This equation can be used in capillary electrophoresis (CE) to design separation systems, and to study interactions between analytes and additives. By using individual capacity factors for each analyte species present in the system, and with the knowledge of the characteristics of each interaction, one can predict the analyte migration behavior in complicated CE systems, including systems with multiple 1:1 interactions and/or higher order interactions.
On‐line competitive immunoassay based on capillary electrophoresis applied to monitoring insulin secretion from single islets of Langerhans
Authors: Tao Li , Aspinwall Craig A. , Kennedy Robert T.
Published: May 2005

An automated on‐line competitive immunoassay based on capillary electrophoresis (CE) was utilized to monitor secretion of insulin from single islets of Langerhans stimulated by glucose and tolbutamide. In the instrument, fluorescein isothiocyanate‐labeled insulin (FITC‐insulin), monoclonal anti‐insulin and perifusate of single islets were mixed on‐line while islets were exposed to different levels of glucose and tolbutamide. Insulin released from single islets competed with FITC‐insulin for antibody binding sites. Therefore, the amounts of bound and free FITC‐insulin were modulated by insulin released from islets. The bound and the free FITC‐insulin were separated by CE every 3 s and the bound over free ratio (B/F) was measured. Insulin levels were obtained by comparing B/F with calibration curves obtained under the same conditions except that the islet perfusate was replaced with various concentrations of insulin. Patterns of insulin secretion stimulated by glucose and tolbutamide observed were comparable to what has been seen previously using radioimmunoassay or enzyme‐linked immunoassay. This on‐line competitive immunoassay system provided a fast and direct way to measure insulin release from single islets. The effects of temperature on antibody‐antigen reaction rate and binding equilibrium were also studied.
Affinity capillary electrophoresis investigation of an epitope on human immunodeficiency virus recognized by a monoclonal antibody
Authors: Qian Xiao‐Hong , Tomer Kenneth B.
Published: May 2005

Affinity capillary electrophoresis (ACE) has been used to investigate the epitope on the human immunodeficiency virus (HIV) core protein p24 recognized by the monoclonal antibody (mAb) 13‐102‐100. The affinity of a series of peptides with N‐ and C‐terminal truncations of the epitope sequence determined by mass spectrometry was studied. The peak area change assay was used for the study of the interactions of the mAb with those peptides, exhibiting tight binding to the mAb, and the migration time shift assay was used to probe the relative affinities of peptides showing weak binding to the mAb. The experimental results show that the monoclonal antibody 13‐102‐100 recognizes the peptide VHPVHAGPIAP with highest affinity. Smaller peptides incorporating only part of the epitope, however, are recognized to some extent in the ACE experiments.
Affinity capillary electrophoresis employing immobilized glycosaminoglycan to resolve heparin‐binding peptides
Authors: VanderNoot Victoria A. , Hileman Ronald E. , Dordick Jonathan S. , Linhardt Robert J.
Published: May 2005

A new capillary electrophoresis technique has been developed for the affinity resolution of synthetic heparin‐binding peptides using an immobilized glycosaminoglycan. Heparin and heparan sulfate were immobilized onto fused silica capillaries using biotin‐neutravidin conjugation. These capillaries exhibited markedly reduced electroosmotic flow and were able to distinguish peptides based on the heparin binding domain of acidic fibroblast growth factor (residues 125–144, GLKKNGSCKRGPRTHYGQKA) that differed only in the stereochemistry of the proline amino acid residue. The peptide based on the native sequence was retarded compared to the peptide having unnatural stereochemistry, consistent with its stronger interaction for immobilized glycosaminoglycan. Improved resolution is also obtained for additional arginine and lysine containing heparin‐binding peptides.
Cloning of prokaryotic genomes in yeast artificial chromosomes: Application to the population genetics of Pseudomonas aeruginosa
Authors: Heuer Thomas , Bürger Christiane , Maaß Giinter , Tümmler Burkhard
Published: May 2005

Yeast artificial chromosomes (YACs) can accommodate large inserts and hence should be attractive tools for intra‐ and interspecies comparisons of bacterial genomes. YAC libraries were constructed from size‐selected partial digests of human and Pseudomonas aeruginosa PAO DNA and SpeI‐restricted PAO DNA. Whereas YACs from human DNA had an average size of 350 kilobase pairs (kbp), a P. aeruginosa sequence larger than 120 kbp was absent or truncated in the eukaryotic host. Coligation occurred for YACs smaller than 40 kbp, but stable YACs with 40–120 kbp large inserts of P. aeruginosa DNA were obtained in high yield. SpeI‐restricted chromosomes from 97 P. aeruginosa strains representing 47 genotypes were hybridized with stable YACs from three equidistant regions of the PAO genome. The low complexity of hybridizing bands demonstrated that the analyzed 100 kbp sequence contigs were stably maintained in most P. aeruginosa isolates from both disease and environmental habitats.
Differential genome analysis of bacteria by genomic subtractive hybridization and pulsed field gel electrophoresis
Authors: Schmidt Karen D. , Schmidt‐Rose Thomas , Römling Ute , Tümmler Burkhard
Published: May 2005

A comprehensive analysis of the differences between the genomes of two closely related bacterial strains should give insight into the molecular basis of their individual phenotypic and genotypic characteristics. Here we present an integrative approach including two different strategies for the thorough investigation of genomic divergence. We have combined two techniques including genomic subtractive hybridization and comparative genome mapping by pulsed field gel electrophoresis (PFGE) techniques. The subtractive method for which a protocol is given herein results in the production of a library of specific DNA sequence tags present only in one strain, while the construction of macrorestriction maps of the bacterial chromosomes yields data about the overall genome organization and the arrangement and distance of gene loci. Comparison of the physical and genetic maps and determination of the map positions of the strain‐specific DNA sequences reveals gross chromosomal modifications, insertions or deletions of additional genetic material, and transpositional events. The further investigation of the strain‐specific regions yields information about the nature and origin of the acquired DNA and their influence on the evolution of the individual bacterial genome. The two methods were applied to differential genome analysis of clonal divergence in Pseudomonas aeruginosa choosing two clone C isolates from diverse habitats.
Low molecular weight proteins: A challenge for post‐genomic research
Authors: Rudd Kenneth E. , Humphery‐Smith Ian , Wasinger Valerie C. , Bairoch Amos
Published: May 2005

The EcoGene project involves the examination of Escherichia coli K‐12 DNA sequences and accompanying annotation in the public databases in order to refine the representation and prediction of the entire set of E. coli K‐12 chromosomally encoded protein sequences. The results of this ongoing effort have been deposited in the SWISSPROT protein sequence database as sequencing of the E. coli genome has progressed to completion in recent years. Through this continuing research, we have discovered that the prediction of low molecular weight (small) proteins, arbitrarily defined as protein sequences ≤ 150 amino acids (aa) in length, is problematic and requires special attention. We describe the small protein subset of EcoGene and the approach used to derive this subset from the complete E. coli genome sequence and database annotations. These E. coli proteins have helped to identify new small genes in other organisms and to identify conserved residues (motifs) using database searches and multiple alignments. Two thirds of the E. coli small proteins have not been characterized experimentally. The careful application of computer and laboratory methods to the analysis of small proteins is needed for accurate prediction, verification and characterization. The problem of accurate protein sequence identification is not limited to small proteins or to E. coli; these problems are encountered to varying degrees throughout all sequence databases.
Comparison of the genome organization of pathogenic Neisseriae (minireview)
Authors: Bautsch Wilfried
Published: May 2005

Current efforts to completely sequence the meningococcal and gonocococcal genomes raise the question whether the lessons learned from the sequenced strains may be safely extrapolated to other members of these species, or whether, in view of the fact that Neisseriae are highly recombinogenic and exhibit a high degree of horizontal intra‐ and interspecies genetic transfer, only clonespecific conclusions are valid. From the known physical and genetic maps of each of two gonococcal and meningococcal strains, it would appear that both species exhibit a species‐specific conservation in their genetic organization while the interspecies comparison revealed several rearrangements, although still with a high overall similarity. However, these data contrast with other evidence suggesting intra‐species rearrangements, such as the nonconserved I‐CeulI macrorestriction patterns of different meningococcal and other neisserial strains. Since I‐Ceul cuts within the 23S‐rRNA sequence, the restriction pattern should give reliable information on the distribution of rrn loci in the neisserial genomes. Further studies are warranted to answer these questions.
Screening of a highly polymorphic microsatellite for microheterogeneity in human identification
Authors: Miścicka‐Śliwka Danuta , Grzybowski Tomasz , Czarny Jakub , Woźniak Marcin
Published: May 2005

Single‐strand conformation polymorphism (SSCP) analysis combined with automated laser fluorescence detection is proposed as a comprehensive, rapid and sensitive method for screening sequence variation of the human beta‐actin‐related pseudogene (HUMACTBP2). Eleven sequenced alleles representing each type of known sequence variant of HUMACTBP2 locus were studied. Allelic variants of the same size but different sequence structures are easily resolved on the basis of their secondary conformation. Fifty ACTBP2 amplification products previously typed on a denaturing gel were repeatedly examined to determine the utility of SSCP analysis in terms of ease of interpretation and reproduction capabilities of the conformational patterns. Eleven sequenced ACTBP2 allelic variants were used as external conformation standards in polymerase chain reaction (PCR)‐SSCP subtyping. This enabled identification of polymorphism in a particular length variant and therefore consistent discrimination between heterozygous samples appeared identical on denaturing gels. Of five “homozygous” samples, one was shown to be heterozygous for two distinct alleles of the same size but different sequences. Thus, the method provides a unique possibility for detecting false homozygotes. The technique complements both denaturing gel electrophoresis and DNA sequencing in studies on the overall variability of the ACTBP2 locus.
Detection of sequence variation in parasite ribosomal DNA by electrophoresis in agarose gels supplemented with a DNA‐intercalating agent
Authors: Zhu Xing Quan , Chilton Neil B. , Gasser Robin B.
Published: May 2005

This study evaluated the use of a commercially available DNA intercalating agent (Resolver Gold™) in agarose gels for the direct detection of sequence variation in ribosomal DNA (rDNA). This agent binds preferentially to AT sequence motifs in DNA. Regions of nuclear rDNA, known to provide genetic markers for the identification of species of parasitic ascarid nematodes (order Ascaridida), were amplified by polymerase chain reaction (PCR) and subjected to electrophoresis in standard agarose gels versus gels supplemented with Resolver Gold. Individual taxa examined could not be distinguished reliably based on the size of their amplicons in standard agarose gels, whereas they could be readily delineated based on mobility using Resolver Gold‐supplemented gels. The latter was achieved because of differences (approximately 0.1–8.2%) in the AT content of the fragments among different taxa, which were associated with significant interspecific differences (approximately 11–39%) in the rDNA sequences employed. There was a tendency for fragments with higher AT content to migrate slower in supplemented agarose gels compared with those of lower AT content. The results indicate the usefulness of this electrophoretic approach to rapidly screen for sequence variability within or among PCR‐amplified rDNA fragments of similar sizes but differing AT contents. Although evaluated on rDNA of parasites, the approach has potential to be applied to a range of genes of different groups of infectious organisms.
Affinity isoelectric focusing of human serum apolipoprotein B‐containing lipoproteins on agarose gels with cyclodextrins
Authors: Ichimura Mitsuko , Okamoto Motoi , Mori Shuji , Tohge Hiroko , Ochi Koji , Harada Hideo
Published: May 2005

In an attempt to subfractionate apolipoprotein B‐containing lipoproteins (apoB‐Lp), agarose isoelectric focusing (IEF) of apoB‐Lp in human sera was carried out in the presence of two kinds of cyclodextrins (CDs), which have different affinities for cholesterol. ApoB‐Lp in the agarose gels was detected by immunofixation, followed by protein staining. In the presence of both 11.4 mg/mL α‐CD and 3.51 mg/mL β‐CD, but not either one alone, IEF separated a maximum of six apoB‐Lp bands. Electrophoresis failed to separate the apoB‐Lp subfractions under similar conditions with CDs. The patterns of apoB‐Lp in IEF with the CDs appeared to differ in some sera. Since the subfractions of apoB‐Lp can be separated without ultracentrifugation of serum, the present method serves for the screening of apoB‐Lp heterogeneity on native serum lipoproteins.
Mixed‐mode separation of polycyclic aromatic hydrocarbons (PAHs) in electrokinetic chromatography
Authors: Luong John H. T. , Guo Yong
Published: May 2005

A mixed‐mode separation technique has been developed and optimized for the separation of the 16 Environmental Protection Agency (EPA) priority polycyclic aromatic hydrocarbons (PAHs). The procedure utilized two different buffer additives as pseudo‐stationary phases with different selectivities towards the analytes. Sodium dioctyl sulfosuccinate (DOSS) displayed selectivities for PAHs which were somewhat similar to the C18 phase in reversed‐phase high performance liquid chromatography (HPLC). High acetonitrile content required for an effective separation prevented the formation of micelles as confirmed by fluorescence spectroscopy. Consequently, the separation could be attributed to the solvophobic association of the PAH molecules with hydrophobic chains of the DOSS surfactant. In another mode of separation, sulfobutylether‐β‐cyclodextrin (SB‐β‐CD) separated the 16 PAHs on the formation of inclusion complexes with the PAHs, and exhibited different selectivities for the PAHs compared to DOSS. SB‐β‐CD and DOSS were then combined in the running buffer to form a mixed pseudo‐stationary phase for the separation of the 16 PAHs. Due to the different selectivities of SB‐β‐CD and DOSS for the PAHs, the separation of the 16 PAHs was appreciably improved compared to that using DOSS or SB‐β‐CD alone. All the 16 PAHs were baseline‐resolved using an optimized running buffer containing 22.5 mM DOSS, 15 mM SB‐β‐CD, 15% acetonitrile and 5 mM hydroxypropyl‐β‐cyclodextrin in 6 mM borate at pH 9.
Molecular size and net charge of pathogenesis‐related enzymes from barley (Hordeum vulgare L., v. Karat) infected with Drechslera teres f. teres (Sacch.) Shoem
Authors: Rothe Gunter M. , Welschbillig Nora , Reiss Ernst
Published: May 2005

Molecular size and net charge of isoforms of pathogenesis‐related (PR) chitinase, β‐1,3‐glucanase and peroxidase were studied in uninfected barley (Hordeum vulgare L., ν. Karat) leaves and in barley leaves infected with the pathogenic fungus Drechslera teres f. teres (Sacch.) Shoem. Molecular characteristics were determined by time‐dependent polyacrylamide gradient gel electrophoresis under native conditions and by applying an extended version of the computer program MOL‐MASS (Rothe, G. M., Weidmann, H., Electrophoresis 1991, 12, 703–709). Uninfected barley leaves contained predominantly one peroxidase isozyme but also three very weak peroxidases. Activities of all of these three peroxidases increased considerably after infection with Drechslera teres. The molecular masses of peroxidases 1 and 3 were estimated to be 38 ± 5 and 42 ± 7 kDa and their apparent valences at pH 8.4 were Z = 3.13 and 3.20, respectively. Amongst the chitinase isoforms, chitinase 1 and chitinase 2 appeared after infection, while chitinase 3 was also observed in uninfected leaves of barley. The molecular mass of chitinase 3 (31 ± 6 kDa; f/fo = 1.20) was larger than that of chitinase 1 (20 ± 2 kDa; f/fo = 1.04) and chitinase 2 (23 ± 3 kDa; f/fo = 1.06). The valence of constitutive chitinase 3 (Z = 1.44 ± 0.81) at pH 8.4 was lower than that of adaptive chitinase 1 (Z = 3.27 ± 1.02) and chitinase 2 (Z = 2.96 ± 1.38). Infection of barley leaves with Drechslera teres also induced the hydrolytic enzyme β‐1,3‐glucanase 1; β‐1,3‐glucanase 2 appeared in uninfected and in infected leaves. Constitutive β‐1,3‐glucanase 2 was smaller (molecular mass 19 ± kDa; f/fo = 1.05) than adaptive β‐1,3‐glucanase 1 (molecular mass 26 ± 4 kDa; f/fo = 1.07). The valence of adaptive β‐1,3‐glucanase 1 (Z = 9.58 ± 4.17) was approximately threefold that of β‐1,3‐glucanase 2 (Z = 2.80 ± 0.93).
Alterations in the expression of P2X1 receptors in failing and nondiseased human atria
Authors: Berry Desiree , Yao Mu , Barden Julian A. , Balcar Vladimir J. , Hansen Mitchell A. , Bennett Maxwell R. , Keogh Anne , dos Remedios Cristobal G.
Published: May 2005

This is the first report of the analysis of the ATP‐specific P2X1 receptor subunit in human hearts. We have examined homogenate samples of human left atria for the presence of P2X1 receptors using Western blots. Anti‐P2X1 immunoreactivity was detected in populations of nondiseased atria as well as in atria from explanted hearts from patients with terminally failing heart conditions such as dilated cardiomyopathy. At least three groups of P2X1 immunoreactive proteins were detected in the Western blots with approximate molecular mass values of 50, 70, and 160 kDa. We report changes in expression of their 50 and 70 kDa components. These changes may be related to the type of deficit in these hearts since the changes have been observed in hearts with decreased ejection fractions characteristic of dilated cardiomyopathy.
Peptide mass fingerprint sequence coverage from differently stained proteins on two‐dimensional electrophoresis patterns by matrix assisted laser desorption/ionization‐mass spectrometry (MALDI‐MS)
Authors: Scheler Christian , Lamer Stephanie , Pan Zaoming , Li Xin‐Ping , Salnikow Johannes , Jungblut Peter
Published: May 2005

Identification of proteins separated by two‐dimensional electrophoresis (2‐DE) is a necessary task to overcome the purely descriptive character of 2‐DE and a prerequisite to the construction of 2‐DE databases in proteome projects. Matrix assisted laser desorption/ionization‐mass spectrometry (MALDI‐MS) has a sensitivity for peptide detection in the lower fmol range, which should be sufficient for an analysis of even weakly silver‐stained protein spots by peptide mass fingerprinting. Unfortunately, proteins are modified by the silver staining procedure, leading to low sequence coverage. Omission of glutaraldehyde increased the sequence coverage, but this improved sequence coverage is still clearly below the sequence coverage starting with Coomassie Brilliant Blue (CBB) R‐250‐stained spots. Other factors additionally seem to modify proteins during silver staining. By decreasing the protein amount, the advantage of very sensitive detection on the gel is lost during identification, because the resulting low sequence coverage is not sufficient for secure identification. Low‐quantity proteins can be identified better starting with CBB G‐250 or Zn‐imidazol‐stained proteins. In contrast, for high‐quantity CBB R‐250‐stained spots, a sequence coverage of up to 90% can be obtained by using only one cleaving enzyme, and up to 80% was reached for medium‐quantity spots after combination of tryptic digest with Asp‐N‐ and Glu‐C digest.
Rapid identification of comigrating gel‐isolated proteins by ion trap‐mass spectrometry
Authors: Arnott David , Henzel William J. , Stults John T.
Published: May 2005

In the search for novel nuclear binding proteins, two bands from a sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) gel were analyzed and each was found to contain a number of proteins that subseqently were identified by tandem mass spectrometry (MS/MS) on a quadrupole ion trap instrument. The bands were digested with trypsin in situ on a polyvinylidence difluoride (PVDF) membrane following electroblot transfer. Analysis of a 2.5% aliquot of each peptide mixture by matrix assisted laser desorption/ionization‐mass spectrometry (MALDI‐MS) followed by an intial database search with the peptide mases failed to identify the proteins. The peptides were separated by reversed‐phase capillary high performance liquid chromatography (HPLC) in anticipation of subsequent Edman degradation, but mass analysis of the chromatographic fractions by MALDI‐MS revealed multiple, coeluting peptides that precluded this approach. Selected fractions were analyzed by capillary HPLC‐electropspray ionization‐ion trap mass spectrometry. Tandem mass spectrometry provided significant fragmentation from which full or partialsequence was deduced for a number of peptides. Two stages of fragmentation (MS3) were used in one case to determine additional dequence. Database searches, each using a single peptide mass plus partial sequence, identified four proteins from a single electrophoretic band at 45 kDa, and four proteins from a second band at 60 kDa. Many of these proteins were derived from human keratin. The protein identifications were corroborated by the presence ofmultiple matching peptide masses in the MALDI‐MS spectra. In addition, a novel sequence, not found in protein or DNA databases, wes determined by interpretation of the MS/MS data. These results demonstrate the power of the quadrupole ion trap for the identification of multiple proteins in a mixture, and for de novo determination of peptide sequence. Reanalysis of the fragmentation data with a modified database searching algorithm showed that the same sets of proteins were identified from a limited number of fragment ion masses, in the absence of mass spectral interpretation or amino acid sequence. The implications for protein identification solely from fragment ion masses are discussed, including advantages for low signal levels, for a reduction of the necessary interpretation expertise, and for increased speed.
Routine identification of proteins from sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) gels or polyvinyl difluoride membranes using matrix assisted laser desorption/ionization‐time of flight‐mass spectrometry (MALDI‐TOF‐MS)
Authors: Fernandez Joseph , Gharahdaghi Farzin , Mische Sheenah M.
Published: May 2005

As the resource laboratory for Rockefeller University our emphasis continues to be on methodology development for the routine analysis of low abundance proteins isolated from native sources. In the past ten years, gel electrophoresis of proteins has become the method of choice for the preparation of microgram and submicrogram quantities of protein for primary structural characterization, and over 95% of the samples submitted for protein identification are either in a gel or bound to polyvinyl difluoride membranes (PVDF). As such, we employ multiple microanalytical approaches encompassing Edman sequence degradation, amino acid and matrix assisted laser desorption/ionization‐time of flight (MALDI‐TOF) mass spectrometric analysis to provide an intergrated protein characterization of such samples. Here we describe the two major services we employ when providing protein identification from in‐gel or PVDF‐bound proteins.
Recycling isoelectric focusing and isotachophoresis
Authors: Bier Milan
Published: May 2005

Of all electrophoretic methods, isoelectric focusing offers the highest resolution and is best suited for preparative applications. Over the years, several instruments were developed for this purpose, all operating in free fluids, in the absence of gels or other supporting matrices. In such systems, the avoidance of gravity or electrically driven convections is essential. Successful stratagems for fluid control included rapid recycling or rotation, in combination with either fine porosity screens or narrow gaps between parallel plates. The most successful apparatus so far is the Rotofor, in which fluid is stabilized by combining horizontal rotation with fine porosity screen partitioning. Recycling isotachophoresis offers the potential of separating proteins at high concentration. A new concept of tangential electrophoresis is described. To optimize the use of these devices for protein separation, low molecular weight, biologically acceptable buffers of known composition are essential. The buffering system developed for this purpose comprises a series of binary buffers that cover the pH range in steps of 1 pH unit or less. The pH gradient can be custom‐designed and is of remarkable stability in operation.
pH‐Regulated electroretention chromatography: Towards a new method for the separation of proteins according to their isoelectric points
Authors: Korlach Jonas , Hagedorn Rolf , Fuhr Günter
Published: May 2005

The pH‐dependent electroretention behavior of model proteins cytochrome c and ribonuclease A was studied in a hollow fiber arrangement, similar to that used in electrical field‐flow fractionation. Field‐induced immobilization of the proteins at the inner wall of the fiber was a function of the pH adjusted in the solution surrounding it, indicating that the pH inside the fiber lumen, relevant for protein migration, quickly equilibrates to the regulated value outside. A complete separation of the model proteins was achieved. Advantages of the principle as well as prospects for the development of a technique separating more than two protein species according to their isoelectric points are discussed.
Separation of viable from radiation‐induced apoptotic lymphocytes by free‐flow electrophoresis
Authors: Chestkov Valery , Baibakov Boris , Radko Sergey P. , Chrambach Andreas
Published: May 2005

A human lymphocyte population undergoing apoptosis in vitro due to γ‐irradiation was fractionated by free‐flow electrophoresis in triethanolamine – Na‐acetate buffers, containing up to 50 mM NaCl, with pH 6.0, 7.2 and 8.5, made isotonic by addition of sucrose. As shown by a flow cytometric analysis of the eluate, the distribution of apoptotic lymphocytes is shifted to the range of higher electrophoretic mobilities relative to that of viable ones at pH 8.5, yielding cell fractions enriched in apoptotic cells by a factor of 3 to 5. The difference in rates of electrophoretic migration observed at a mildly alkaline pH but not at a neutral or mildly acidic one suggests that the surface of apoptotic lymphocytes is more acidic than that of viable ones.
Lectin‐gradient gel affinity electrophoresis of glycoproteins
Authors: Taketa Kazuhisa , Kamakura Kozue , Fukazawa Masako , Ikeda Satoru , Taga Hiroko
Published: May 2005

Lectin‐gradient agarose gel affinity electrophoresis was developed for identification of glycoforms of glycoproteins in lectin affinity electrophoresis. Gradation of lectin was done by stacking agarose gel blocks with increasing concentrations of lectin (discontinuous system) and by keeping the plate in a moist chamber at 4°C overnight (continuous system) before electrophoresis. On the visualization of separated glycoform lines, the antibody‐affinity blotting was superior for low concentrations of α‐fetoprotein. Fluoresceine isothiocyanate (FITC) labeling of whole serum proteins, enzyme activity staining for alkaline phosphatase, and prestaining for lipoproteins were also applicable for visualization of proteins at higher concentrations. The conventional Western blotting can not be recommended because of the competition between lectin and glycoproteins in binding to nitrocellulose membrane. Lectin‐gradient affinity electrophoresis also had a wide application for optimization of the condition of lectin affinity electrophoresis.
Continuous flow isoelectric focusing for purification of proteins
Authors: Soulet Nathalie , Roux‐de Balmann Hélène , Sanchez Victor
Published: May 2005

To estimate the feasibility of isoelectric focusing in a continuous mode, the conditions for obtaining stable and linear pH gradients were first investigated, using a mixture of carrier ampholytes. Then the operating parameters allowing the isoelectric focusing of bovine serum albumin as model protein were determined. A theoretical approach was used to find conditions minimizing the electrohydrodynamic effect, mainly responsible for protein dispersion, for the separation of bovine serum albumin and α‐lactalbumin from bovine milk.
The effects of electroendosmosis in agarose electrophoresis
Authors: Guo Yaojun , Li Xinhui , Fang Yong
Published: May 2005

The effects of agarose gel strips without and with 0.03 mr electroendosmosis (EEO) on isoelectric focusing (IEF) were studied. It is shown that only agarose without EEO can be used for IEF. The effects of electrode buffer strips using agarose with different EEO of 0, 0.03, 0.08, 0.20 mr on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and native PAGE were also studied. It apparently did not affect SDS‐PAGE, but affected native PAGE to a certain extent. The higher the EEO value was, the more water was present on the agarose gel strip during a separation. Agarose gel strips with an EEO of 0.2 mr are not suitable as electrode buffer strips for native PAGE.
Comparison of different methods to produce single‐strand DNA for identification of canned tuna by single‐strand conformation polymorphism analysis
Authors: Rehbein Hartmut , Mackie Ian M. , Pryde Susan , Gonzales‐Sotelo Carmen , Perez‐Martin Ricardo , Quinteiro Javier , Rey‐Mendez Manuel
Published: May 2005

By using single‐strand conformation polymorphism (SSCP) analysis of three amplicons of the cytochrome b gene obtained by the polymerase chain reaction (PCR) it was possible to differentiate between various species of tunas and bonitos processed as canned fish. Four different techniques were used to produce single‐strand DNA (ssDNA): (i) Denaturation of double‐strand DNA (dsDNA) by formamide and alkali, (ii) two‐step asymmetrical PCR, (iii) one‐step asymmetrical PCR, and (iv) exonuclease digestion of the phosphorylated strand of dsDNA. The technique rendering optimal results depended on the type of amplicon (i.e. the sequence).
The combination of Indian ink staining with immunochemiluminescence detection allows precise identification of antigens on blots: Application to the study of glycosylated barley storage proteins
Authors: Eynard Lucia , Laurière Michel
Published: May 2005

The localization after blotting of specific spots in two‐dimensional electrophoretic protein pattern was achieved using, in that order, Indian ink protein staining and immunodetection with chemiluminescence on the same membrane. Indian ink did not inhibit significantly the antibody reactions even after overnight staining. It produces permanent staining that did not quench the chemiluminescent signal, recorded on a film. This allowed perfect matching between the specific and the total protein patterns. The procedure was applied to the identification of glycoproteins present in barley storage protein preparations.
Proteins of rat serum: II. Influence of some biological parameters of the two‐dimensional electrophoresis pattern
Authors: Miller Ingrid , Haynes Paul , Gemeiner Manfred , Aebersold Ruedi , Manzoni Cristina , Lovati Maria Rosa , Vignati Mara , Eberini Ivano , Gianazza Elisabetta
Published: May 2005

This report complements the database already detailed for serum proteins of healthy adult male rats (P. Haynes et al., Electrophoresis 1998, 19, 1484–1492). The influence on the two‐dimensional electrophoresis (2‐DE) pattern of certain physiological conditions (sex, age) was studied as well as of changes in thyroid metabolism. We have extended the information about the major components of rat serum by identifying the proteins typical for the response to acute inflammation. Analyzing 27 spots, six proteins not found in control sera could be recognized; migration at overlapping or close positions with five already characterized proteins was observed for some. A compilation of all our rat data can be accessed through: http://weber.u.washington.edu/∼ruedilab/aebersold.html.
Highly cationic anti‐DNA antibodies in patients with lupus nephritis analyzed by two‐dimensional electrophoresis and immunoblotting
Authors: Kohro‐Kawata Junko , Wang Pei , Kawata Yasunobu , Matsuzaki Masunori , Nakamura Kazuyuki
Published: May 2005

The relationship between chemical properties of anti‐DNA antibodies (Abs) and lupus nephritis was investigated. The anti‐DNA Abs in sera from systemic lupus erythematosus (SLE) patients were separated by two‐dimensional electrophoresis (2‐DE) and immunoblotting with goat anti‐human IgG Abs. Highly cationic anti‐DNA Abs were detected in deoxyribonuclease I (DNase I)‐treated sera from patients with lupus nephritis (in 8 of 9 cases) but not in the sera from SLE patients without nephritis (in 0 of 9 cases), normal subjects, or patients with other renal diseases (in 0 of 7 cases). The mean titers of anti‐dsDNA Abs in patients with lupus nephritis were not significantly different from those in SLE patients without nephritis. The highly cationic anti‐DNA Abs in the sera disappeared after incubation with heparin‐Sepharose. These results suggest that highly cationic anti‐DNA Abs are specific for lupus nephritis and may be involved in development of lupus nephritis via the binding to glycosaminoglycans on the endothelial cell surface.
In memory of Harry Rilbe: An outstanding scientist who made many valuable contributions to the development of electrophoretic methods
Authors: Vesterberg Olof
Published: May 2005

In July 1997 Professor Harry Rilbe passed away at the age of 84. Harry Rilbe was an outstanding personality skilled in chemistry, optics and mathematics, and he will be remembered for his important contributions to the development of separation methods, notably moving boundary electrophoresis and isoelectric focusing. The latter method is used for analytical and preparative purposes in thousands of laboratories worldwide. Isoelectric focusing is indispensable for characterization of proteins from humans, animals, plants and microorganisms. Isoelectric focusing is thus useful for the understanding of the function of genes and, accordingly, for progress in the life sciences. A biographical retrospect, “A scientific life with chemistry, optics and mathematics”, was published by Harry Rilbe in Electrophoresis 1984, 5, 1–17.
Theory and practice of isoelectric focusing of interacting systems
Authors: Cann John R.
Published: May 2005

The theory of mass transport coupled to reversible protein interactions forms the basis for computer simulation of the isoelectric focusing behavior of several model systems. These include pH‐dependent conformational transition, carrier ampholyte‐induced interactions and protein‐ligand interactions. The computational results compare favorably with experimental observations. In addition, a method is formulated for an isoelectric focusing procedure which enables determination of intrinsic ligand‐binding constants for statistical binding of a charged ligand, binding to heterogeneous sites, and cooperative binding.