bluuebunny/crossref_metadata_2025 huggingface dataset:
The human keratinocyte two‐dimensional protein database (update 1994): Towards an integrated approach to the study of cell proliferation, differentiation and skin diseases
Authors: Celis Julio E. , Rasmussen Hanne Holm , Olsen Eydfinnur , Madsen Peder , Leffers Henrik , Honoré Bent , Dejgaard Kurt , Gromov Paul , Vorum Henrik , Vassilev Alex , Baskin Yasemin , Liu Xiangdang , Celis Ariana , Basse Bodil , Lauridsen Jette B. , Ratz Gitte P. , Andersen Annette H. , Walbum Else , Kjærgaard Inge , Andersen Inger , Puype Magda , Van Damme Josef , Vandekerckhove Joël
Published: May 2005

The master two‐dimensional (2‐D) gel database of human keratinocytes currently lists 3087 cellular proteins (2168 isoelectric focusing, IEF; and 919 nonequilibrium pH gradient electrophoresis, NEPHGE), many of which correspond to posttranslational modifications. 890 polypeptides have been identified (protein name, organelle components, etc.) using one or a combination of procedures that include (i) comigration with known human proteins, (ii) 2‐D gel immunoblotting using specific antibodies (iii) microsequencing of Coomassie Brilliant Blue stained proteins, (iv) mass spectrometry and (v) vaccinia virus expression of full length cDNAs. These are listed both in alphabetical order and with increasing SSP number, together with their Mr, pI, cellular localization and credit to the investigator(s) that aided in the identification. Furthermore, we list 239 microsequenced proteins recorded in the database. We also report a database of proteins recovered from the medium of noncultured, unfractionated keratinocytes. This database lists 398 polypeptides (309 IEF; 89 NEPHGE) of which 76 have been identified. The aim of the comprehensive databases is to gather, through a systematic study of keratinocytes, qualitative and quantitative information on proteins and their genes that may allow us to identify abnormal patterns of gene expression and, ultimately, to pinpoint signaling pathways and components affected in various skin diseases, cancer included.
REF52 on global gel navigator: An internet‐accessible two‐dimensional gel electrophoresis database
Authors: Boutell Thomas , Garrels James I. , Franza B. Robert , Monardo Pat J. , Latter Gerald I.
Published: May 2005

Until recently, the REF52 2‐D gel database of experiments with rat cell lines was accessible only with special software. This database has now been made available to all investigators with access to the Internet, using the World Wide Web (WWW) technology. The package which delivers the database through the WWW has been named the Global Gel Navigator and can be used to explore the data by several methods, including the direct selection of proteins in the displayed gel using the mouse.
Detection of point mutations by capillary electrophoresis in liquid polymers in temporal thermal gradients
Authors: Gelfi Cecilia , Righetti Pier Giorgio , Cremonesi Laura , Ferrari Maurizio
Published: May 2005

A new and fast method is described for detection of point mutations in polmerase‐chain reaction (PCR)‐amplified DNA, based on capillary electrophoresis in sieving liquid polymers in presence of temporal thermal gradients. The background electrolyte contains a constant amount of denaturing agent (e.g., 6 M urea) and the DNA fragments are injected in a constant‐temperature plateau below the melting temperature (Tm). After loading, a temperature ramp is activated (typically from 0.2 to 0.6°C/min, according to the melting profiles of the DNA duplexes under investigation) with resultant branching of homo‐ and heteroduplexes at different times along the migration path. In the case of individuals heterozygous for a point mutation, the expected four‐band pattern is obtained. The temperature gradient is not produced externally, via circulating coolant and a thermostat, but is generated internally by using a dedicated computer program able to calculate the precise inner temperature under given electric conditions. The method is applied to the identification of three point mutations located in exo 17b (R1066H, R 1066C, F1052V) and of two polyrmorphisms located in exon 14a (V868V, T854T) of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.
Consequences of a maximum existing in the dependence of separation selectivity on concentration of cyclodextrin added as chiral selector in capillary zone electrophoresis
Authors: Vespalec Radim , Fanali Salvatore , Boček Petr
Published: May 2005

The maximum in the dependence of the separation selectivity on the concentration of cyclodextrin may be utilized for the determination of the mean value of host‐guest interaction constants of the separated enantiomers. For the hosting of N‐t‐BOC‐DL‐tryptophan by β‐cyclodextrin, the mean value of these constants, found by drop line estimated from the cyclodextrin concentration corresponding at the maximum, is 350 L.mol−1. The separation at this concentration of cyclodextrin offers the highest resolution in the shortest separation time. It is shown that commonly used simple preliminary experiments, testing the capability of a cyclodextrin to resolve chiral compounds, and based on relatively high cyclodextrin concentrations, may easily lead to incorrect conclusions in cases of enantiomers strongly interacting with the cyclodextrin used.
Capillary zone electrophoresis for the rapid screening of banned drugs in sport
Authors: Gonzalez Esther , Laserna Jose J.
Published: May 2005

Capillary zone electrophoresis was used to analyze some drugs illegally used in sports. The spectroscopic characteristics and electrophoresis migration parameters of diuretics, narcotics‐analgesics and β‐blockers were established. Analytical figures of merit including parameters on the precision of migration and limits of detection are discussed. Results for the separation of drugs of different doping families are discussed. An analysis of urine from a patient receiving daily dosis of atenolol is presented.
Modulation of mts1 expression in mouse and human normal and tumor cells
Authors: Grigorian Mariam , Tulchinsky Eugene , Burrone Oscar , Tarabykina Svetlana , Georgiev Georgii , Lukanidin Eugene
Published: May 2005

The mts1 gene, encoding small Ca2+‐binding protein of the S100‐family, is considered as a gene whose activity correlates with the manifestation of a metastatic phenotype of tumor cells. It was shown before that the mts1 is expressed not only in metastatic tumor cells but also in some normal tissues, namely in so‐called “lymphoid” organs: spleen, thymus; bone marrow. In this work we analyzed in more detail the expression of mts1 in human and mouse hematopoietic cells and cell lines. A high level of mts1 RNA was observed in T‐lymphocytes, neutrophils, monocytes/macrophages and in corresponding cell lines. Controversially, the mts1 gene was silent in B‐lymphocytes as well as in myeloma and erythroleukemia cell lines. The possibility of modulating the mts1 gene expression by the action of different agents was demonstrated. Mitogens, such as lipopolysaccharides (LPS), interferon (IFNγ), and concanavalin A (Con A), modulate the level of the mts1 gene expression in hematopoietic cells differently. Calcium ionophore, A23187, can also be regarded as a modulator of the mts1 gene expression, since its addition to the cells results in a substantial decrease of the mts1 RNA level. It was shown that the mts1 RNA's half‐life is relatively long, more than 24 h. We therefore believe that calcium ionophore can activate some ribonucleases which degrade the mts1 RNA. Cycloheximide prevents the effect of A23187 and stabilizes the mts1 RNA, probably by blocking the synthesis of these nucleases. Thus, the obtained data indicate that the agents which are capable of changing the physiological status of the cells also modulate the mts1 gene expression.
Several small GTP‐binding proteins are strongly down‐regulated in simian virus 40 (SV40) transformed human keratinocytes and may be required for the maintenance of the normal phenotype
Authors: Gromov Pavel S. , Celis Julio E.
Published: May 2005

High resolution two‐dimensional (2‐D) gel electrophoresis in combination with the blot overlay nucleotide binding assay was used to reveal low molecular weight GTP‐binding proteins expressed by primary cultured, normal human keratinocytes. Forty one small GTP‐binding proteins (30 isoelectric focusing, IEF; and 11 nonequilibrium pH gradient electrophoresis, NEPHGE) ranging in molecular weights from 18000 to 30000 and isoelectric points from 4.4 to 8.0 were detected and mapped in the master human keratinocyte database. Four GTP‐binding proteins were identified by 2‐D gel immunoblotting and these correspond to rap 1 and 2 and two forms of rab6. ras Proteins are most likely present in the [α32P]GTP 2‐D gel blots but their levels may be too low to be detected by immunoblotting. Quantitative changes in the relative expression levels of [α32P]GTP‐binding proteins in normal proliferating and simian virus 40 (SV40) transformed human keratinocytes (K 14) were determined by scintillation counting of the radioactive spots excised from the nitrocellulose blots. The results showed that thirteen of these proteins were not expressed in transformed K14 keratinocytes, implying that they may play a role in the maintenance of the normal cell phenotype.
Apoptotic cell death analyzed at the molecular level by two‐dimensional gel electrophoresis
Authors: Robaye Bernard , Døskeland Anne Paulus , Suarez‐Huerta Nathalie , Døskeland Stein Ove , Dumont Jacques E.
Published: May 2005

The pattern of protein expression and phosphorylation after an apoptotic stimulus has been studied in two systems. Bovine aortic endothelial cells were induced to undergo apoptotic cell death by a combination of a cytokine (tumor necrosis factor, TNF) and inhibitors of protein synthesis, like cycloheximide. Two‐dimensional (2‐DE) electrophoresis of proteins from such cells revealed specific proteolysis of distinct proteins, some at an early stage of apoptosis and some at a later stage. These proteins may have antiapoptotic properties. In rat IPC‐81 promyelocytic leukemia cells, cAMP induced apoptosis. 2‐DE of such cells pulse‐labeled with [35S]methionine revealed two “novel” protein spots (of 30 kDa and 46 kDa, respectively), induced very rapidly by a posttranscriptional mechanism. It is proposed that “dysphosphorylation” may accompany apoptosis in general, since both endothelial cells treated with TNF/cycloheximide and IPC‐81 cells treated with cAMP analog or the apoptosis‐inducing phosphatase inhibitors okadaic acid or calyculin A all showed altered protein phosphorylation patterns, as revealed by 2‐DE electrophoresis of proteins from cells prelabeled with 32Pi.
Gel electrophoretic analysis of nuclear matrix fractions isolated from different human cell lines
Authors: Kallajoki Markku , Osborn Mary
Published: May 2005

The nuclear matrix is operationally defined as the structure that remains after nuclei are extracted with nonionic detergent and with high salt and are digested with nucleases. Thus the nuclear matrix protein composition is critically dependent on the isolation conditions. We have compared nuclear matrices isolated from human cell lines by two different methods. First, isolated nuclei were extracted as above to obtain a matrix fraction. This method showed a substantial contamination by cytoplasmic intermediate filaments but immunization of mice resulted in antibodies recognizing nuclei and the mitotic spindle apparatus. Second, a nuclear matrix fraction was made by extracting whole cells as above and dissolving the residue in urea and dialysing against an assembly buffer to precipitate intermediate filament proteins (Fey, E. G. and Penman, S., Proc. Natl. Acad. Sci. USA 1988, 85, 121–125). Such fractions showed complex protein patterns in silver‐stained two‐dimensional gels for four cell lines: HeLa, MCF‐7, SW13 and the U333CG/343MG glioma line. While some proteins in the nuclear matrix fraction were common to all cell lines, others appeared cell‐line specific. Two‐dimensional gels and the immunoresponse in mice again showed contamination of these preparations with cytoplasmic proteins. These results clearly show the difficulties associated with protein chemical analysis of nuclear matrices: the preparations have substantial cytoplasmic contamination, the polypeptide composition is extremely complex and the yield of individual polypeptides is low. Thus, without further experiments one cannot say which proteins are true nuclear matrix components. In addition, our results underline the value of dissecting the nuclear matrix by analyzing its individual components with monoclonal antibodie, and we show examples of the nuclear staining patterns of three different classes of monoclonal antibodies isolated by this approach.
Separation and analysis of pig pancreatic zymogen granules with free flow electrophoresis and lectins
Authors: Spaans M. Carina , Tobler Markus , Ammann Rudolf W. , Freiburghaus Andreas U.
Published: May 2005

Purified pig pancreatic zymogen granules were subjected to free flow electrophoresis (FFE) in an acetate buffer system (acetic acid/NaOH, pH 5.5) to detect the presence or absence of more than one population or zymogen granules. Pig pancreatic zymogen granules were purified by differential and density gradient centrifugation and subjected to FFE. Fractions were analyzed for protein, α‐amylase (EC 3.2.1.1) and 5′‐nucleotidase (EC 3.1.3.5) as marker enzymes for zymogen granule content and membranes, respectively. Only one distinct peak, with coincident α‐amylase and 5′‐nucleotidase activity, and most protein was detected, which reflects the presence of a single population of intact zymogen granules. This was confirmed by electron microscopy. When the granules were incubated with different lectins before FFE, the one distinct peak representing intact zymogen granules was shifted towards the cathode in the case of concanavalin A (Con A) and Ricinus communis agglutinin 120 (RCA 120). No splitting of the peak occurred. Our results do not support the hypothesis of a coexistence of more than one distinct population of zymogen granules.
Specific, sensitive and accurate quantification of albumin, retinol binding protein and transferrin in human urine and serum by zone immunoelectrophoresis assay (zia)
Authors: Vesterberg Olof
Published: May 2005

For zone immunoelectrophoresis assay (ZIA) glass. tubes, ID 2 mm and 90 mm high, are filled to 2/3 with buffer containing agarose and antibodies against the protein to be quantified, each sample being pipetted on top of separate agarose gel rods. On electrophoresis at 35–150 V for several hours, the sample proteins enter the gel with resultant immunoprecipitates, visualized by staining. The extension of each immunoprecipitation zone from the upper gel surface (measured with a ruler) is directly proportional to the amount of protein in each sample and can easily be quantitated by comparison with a linear calibration curve. ZIA can be used for quantification of several proteins in blood serum and plasma as well as in urine, as is illustrated for albumin, retinol‐binding protein (RBP) and transferrin. The recovery of the pure proteins added to urine is often close to 100%. ZIA has many advantages: (i) simple apparatus and procedure (no gel punching nor cooling), (ii) minimal antiserum consumption (1 mL may allow > 1000 assays), (iii) electrophoresis can be performed within a few hours or overnight, (iv) low coefficient of variation (often < 4%), (v) linear calibration curves, (vi) low detection limit (< 20 ng/mL), (vii) wide concentration ranges, (viii) no kits nor unique antisera preparation are required, and (ix) good agreement with the results from other methods.
Capillary zone electrophoresis of polymerase chain reaction‐amplified DNA fragments in polymer networks: The case of GATT microsatellites in cystic fibrosis
Authors: Gelfi Cecilia , Orsi Antonia , Righetti Pier Giorgio , Brancolini Valeria , Cremonesi Laura , Ferrari Maurizio
Published: May 2005

In cystic fibrosis (CF), the most common mutation, delta F508 (a three‐base‐pair deletion) accounts for ca. 70% of mutations in the worldwide population. The majority of other mutations (more than 350 reported so far to the Genetic Analysis Consortium) have been detected in single cases, thus rendering quite cumbersome a molecular diagnostic approach for the identification of CF chromosomes. As an alternative, linkage analysis based on intragenic polymorphism can be useful for prenatal diagnosis and CF‐carrier detection, provided that the heterozygosity of the allelic forms is very high. For this purpose, DNA microsatellites, consisting of two to epta nucleotide repeat clusters, displaying a high degree of polymorphism, are being increasingly used as markers in linkage studies. Two main allelic forms, one hexameric (111 bp) and one heptameric (115 bp), of a tetranucleotide (GATT) repeat polymorphism, at the junction of intron IVS6a and exon 6b, have been amplified by PCR technology. These two alleles can be separated in a 10–20%T polyacrylamide gradient gel and detected by ethidium bromide staining. As an alternate procedure, these two fragments are efficiently separated by capillary zone electrophoresis in a viscous solution of 6%T linear polyacrylamide and detected by their intrinsic absorbance at 254 nm.
Capillary electrophoresis of polymerase chain reaction‐amplified products in polymer networks: The case of Kennedy's disease
Authors: Nesi Marina , Righetti Pier Giorgio , Patrosso Maria Cristina , Ferlini Alessandra , Chiari Marcella
Published: May 2005

In Kennedy disease (an X‐linked motoneuronal disorder associated with an increase in the number of (CAG)n triplet repeats in the first exon of the Androgen receptor gene; about twice as many as in normal conditions), polymerase chain reaction‐amplified genic products exhibit two DNA fragments in the heterozygous female carriers, one with a range between 468 and 495 bp in the normal polymorphic population and a fragment corresponding to the pathological state that reaches 573 bp. These fragments are easily separated by gel‐slab electrophoresis and detected by intercalating dye staining (ethidium bromide). As an alternative procedure, capillary zone electrophoresis in polymer networks, consisting of 8% polyacryloylaminoethoxyethanol at 0% cross‐linker, offers a simple procedure for separation and on‐line detection via UV absorbance at 254 nm, thus avoiding additional staining steps. The capillary column can be repeatedly used for up to 80–100 times and the electropherogram is stored on a magnetic support. Easy comparison among different runs is obtained by aligning all tracings to an internal standard of a 650 bp fragment added as a marker.
Studies of serum protein complexes with nickel using crossed immunoelectrophoresis
Authors: Nielsen Jeanet Løgsted , Poulsen Otto Melchior , Abildtrup Anne
Published: May 2005

Crossed immunoelectrophoresis of human serum spiked with nickel in the range 0.85–24 mmol Ni/L was used to study nickel‐protein complexes. These high concentrations, which are far higher than the physiological level (approximately 7.8 nmol/L), were used to saturate both high and low affinity binding sites. Addition of increasing amounts of nickel resulted in dose‐dependent changes of the electrophoretic patterns of prealbumin, α‐1‐lipoprotein, α‐1‐antitrypsin and α‐2‐macroglobulin. Radioactive 63Ni was used for crossed immunoelectrophoresis autoradiography experiments for further identification of nickel‐protein complexes. When a 63Ni pulse of 740 kBq/application was used, many human serum proteins were labeled. When using a 63Ni pulse of 185 kBq/application only albumin and α‐1‐antitrypsin were visualized clearly. The binding of large amounts of nickel to albumin, visualized by autoradiography, may reflect the high abundance of albumin in human serum as compared to other serum proteins. Addition of nickel to serum proteins resulted in liquid phase precipitation of serum proteins, and rocket immuno‐electrophoresis was used to demonstrate that IgG in particular is precipitated. This precipitation of serum proteins may disturb the elution profile when chromatographic techniques are used to analyze nickel‐protein complexes. Consequently, immunoelectrophoretic methods may also be attractive alternatives to column chromatographic techniques. The present study demonstrated that, besides the nickel‐binding of albumin and α‐2‐macroglobulin, several other serum proteins have nickel‐binding affinity.
N,N,N′,N′‐Tetramethyl‐1,3‐butanediamine as effective running electrolyte additive for efficient electrophoretic separation of basic proteins in bare fused‐silica capillaries
Authors: Corradini Danilo , Cannarsa Gianfranco
Published: May 2005

The effect of N,N,N′,N′‐tetramethyl‐1,3‐butanediamine (TMBD) in the running electrolyte on the electroosmotic flow and the migration behavior of four standard basic proteins in bare fused‐silica capillaries was examined at pH 4.0, 5.5, and 6.5. Depending on the electrolyte pH and additive concentration the electroosmotic flow was either cathodic or anodic. A similar Langmuirian‐type dependence of the electroosmotic flow on the concentration of TMBD in the running electrolyte was found at the three experimented pH values, which may be indicative of the specific adsorption of the additive in the immobilized region of the electric double layer at the interface between the capillary wall and the electrolyte solution. Electrophoretic separations of the four standard basic proteins performed at the three above pH values, showed well‐resolved, efficient and symmetric peaks, demonstrating the utility of this additive for protein electrophoresis in bare fused‐silica capillaries. The variations in separation efficiency, peak capacity, resolution and reproducibility of migration times as a function of the additive concentration at pH 6.5 were also examined.
Capillary electrophoretic separations of biotechnology‐derived proteins in E. coli fermentation broth
Authors: Strege Mark A. , Lagu Avinash L.
Published: May 2005

A capillary electrophoresis (CE) method was developed for the analysis of a recombinant DNA protein in a fermentation broth matrix. A polyacrylamidecoated capillary was employed to eliminate electroosmotic flow, facilitating relatively rapid run times. Selectivity and efficiency were enhanced significantly through the incorporation of magnesium sulfate and acetonitrile, an organic modifier, into the separation buffer. Both of these additives appeared to influence the separation by diminishing the interaction of the protein analyte with the charged micelles present in the buffer. The quantitation capabilities of the CE method were validated and found to be comparable to the standards set by chromatographic techniques previously developed for similar applications.
Quantitative analysis of major whey proteins by capillary electrophoresis using uncoated capillaries
Authors: Recio Isidra , Molina Elena , Ramos Mercedes , de Frutos Mercedes
Published: May 2005

A method that allows separation and quantitation of the main whey proteins by capillary electrophoresis using uncoated capillaries is proposed. Separations are performed using 100 mM borate buffer, pH 8.2, containing 30 mM sodium sulfate. The use of high pH and high ionic strength buffer reduces adsorption of proteins on the capillary wall, making their separation possible. Reproducibility of migration times and areas of peaks are improved by optimizing the capillary equilibration protocol and by using an internal standard. Relative standard deviations ranging between 0.74 and 1.03% for migration times and 2.14 to 5.23% for areas of major peaks are obtained. Detection limits equal to or lower than 0.5 mg/100 mL are achieved. Linear relationships of peak area versus concentration have been used to quantitate bovine serum albumin (BSA), α‐LA (α‐lactalbumin), β‐LG A (β‐lactoglobulin A) and β‐LG B (β‐lactoglobulin B) in cow's milk subjected to different thermal treatments. Electrophoretic profiles of these milk samples show peaks from other peptides besides those from main proteins. Characteristic patterns for whey from different species are obtained.
Temperature‐gradient gel electrophoresis as a screening tool for polymorphisms in multigene families
Authors: Schlötterer Christian
Published: May 2005

Information about sequence variability between different copies of a multigene family is indispensable for understanding the evolutionary mechanisms acting on multigene families. However, their high copy number has been a major obstacle to systematic analysis. Exemplified by the internal transcribed spacer 1 (ITS1) of the rDNA in Drosophila melanogaster, it is shown how temperature gradient gel electrophoresis (TGGE) can be used to study sequence polymorphisms in a multigene family. Experimental conditions influencing the melting behavior of the ITS1 fragment are discussed as well as discrepancies between observed and calculated melting patterns.
Temperature gradient gel electrophoresis for analysis of a polymerase chain reaction‐based diagnostic clonality assay in the early stages of cutaneous T‐cell lymphomas
Authors: Menke Martin A. O. H. , Tiemann Markus , Vogelsang Dörthe , Boie Christina , Parwaresch Reza
Published: May 2005

By means of a multiplex polymerase chain reaction (PCR) we amplified rearranged T‐cell receptor γ chain genes to detect monoclonality in 370 formalin‐fixed skin biopsy specimens, showing histological features of parapsoriasis or mycosis fungoides. PCr products were analyzed by temperature gradient gel electrophoresis (TGGE). We selected 20 positive cases for use in a comparison of this technique with conventional agarose and polyacrylamide gel electrophoresis (PAGE). With TGGE the T‐cells had shown monoclonality in 272 of the 370 cases; with agarose electrophoresis they did so in only 5 of the 20 selected cases and with PAGE in 16. Where multiple biopsy specimens from the same patient were analyzed, PCR products showed identical rearrangement patterns in TGGE. TGGE is an efficient technique that works on routine material and can help to verify a histological diagnosis of cutaneous T‐cell lymphoma.
Effect of pH and sodium dodecyl sulfate concentration on the analytical window in the direct‐injection analysis of plasma samples by capillary electrophoresis
Authors: Wätzig Hermann , Lloyd David K.
Published: May 2005

Plasma samples can be analyzed with direct injection onto a capillary electrophoresis system if interactions between the plasma proteins and capillary walls are minimized. This can be achieved using micellar electrokinetic capillary chromatography with a surfactant such as sodium dodecyl sulfate. The surfactant complexes with the proteins, giving them a net negative charge, and thus causes them to be repelled from the negatively‐charged fused‐silica capillary walls. Migrating as anions, the complexed proteins appear late in the electropherogram. In such analyses, it is important to manipulate the mobility of the analyte(s) of interest such that they migrate in the useful analytical time window before the plasma proteins. In this article, a study of the effect of buffer pH and surfactant concentration on the width of the migration time window is reported. It is shown that at pH 6 the migration time window is wide, but reduced electroosmosis can result in inordinately long run times; pH 7 gives an acceptable analytical window with acceptable run times over a wide range of sodium dodecyl sulfate (SDS) concentrations; pH 8 and higher are most useful when higher concentrations of surfactant (> 50 mM SDS) are employed.
Molecular characterization of Borrelia burgdorferi sensu lato strains by pulsed‐field gel electrophoresis
Authors: Busch Ulrich , Teufel Cecilia Hizo , Boehmer Reinhard , Wilske Bettina , Preac‐Mursic Vera
Published: May 2005

The large restriction fragment patterns (LRFP) and linear plasmid profiles of eight tick isolates of Borrelia burgdorferi sensu lato were investigated with pulsed‐field gel electrophoresis (PFGE). The whole cell lysate was examined with sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). The MluI LRFP differentiates two species of Borrelia burgdorferi sensu lato (Borrelia burgdorferi sensu stricto and Borrelia garinii). LRFP is a suitable method to demonstrate genetic hetero‐ or homogeneity of isolates within one species without subsequent hybridization utilizing diverse probes. Different strains with similar or identical LRFP can be further discriminated by plasmid profile analysis. Our results show that each strain analyzed had a different plasmid profile. Therefore the linear plasmid profile has a potential application as a strain typing procedure. SDS‐PAGE of whole‐cell lysate supports the findings of homology within the B. burgdorferi sensu stricto species and the heterology within the B. garinii species.
Electrophoretic resolution versus fluctuations of the lateral dimensions of a capillary
Authors: Slater Gary W. , Mayer Pascal
Published: May 2005

Because the local electrical resistance is inversely proportional to the local cross‐section of a capillary, the intensity of the electric field varies along the migration path if the inner diameter of the capillary is not constant. Therefore, fluctuations of the lateral dimensions of a capillary can directly affect the net elution time as well as the peak width, and, hence the final resolution. In this article, we develop the theoretical framework for the study of such effects. We then examine the simple case where both the mobility and the diffusion coefficient are field‐independent; in particular, we demonstrate that resolution can be severely reduced if the inner walls are not flat, and that optimal resolution is always obtained for perfectly flat walls. Generalized to ultrathin gels, our results clearly indicate that both random and systematic variations of the gel thickness can greatly affect the performance of the separation process. Acceptable degrees of flatness are estimated for both geometries. This study thus provides a quantitative understanding of the type of quality control one requires to obtain optimal results with capillaries and ultrathin gels.
Dispersion coefficients of a protein and DNA fragment in polyacrylamide gel electrophoresis as a function of parameters defining the effective gel pore size and particle size
Authors: Chang Huan‐Tsung , Chrambach Andreas
Published: May 2005

The dispersion coefficient, D′, of the representative homogeneous protein, conalbumin, decreases linearly as the polyacrylamide concentration increases from 4 to 14%T (2%C), and varies in a biphasic fashion as %C (Bis) is increased from 2 to 20%, with a broad peak between 5 and 15%C. D′ increases linearly with the concentration of the initiator, potassium persulfate, in the range of 0.01–0.15%. D′ remains constant when the field strength is varied from 5 to 15 V/cm. A DNA fragment (1857 bp) exhibits a constant D′ in 4–6% polyacrylamide (2%C) at a field strength of 1 V/cm, and a linearly increasing D′ at 5 V/cm, in analogy to its previously observed behavior in agarose gels. In solutions of uncrosslinked polyacrylamide, the decrease of the D′ of conalbumin with polymer concentration is not significantly different from that in 2% N,N′‐methylenebisacrylamide‐crosslinked gels in the range of 4–14%T, while the decrease of mobility with polyacrylamide concentration is much steeper in 2% crosslinked compared to uncrosslinked polymer. Finally, –∂ (log D′)/∂T was found to be proportional to the retardation coefficient, KR (= –∂ (log μ)/∂T), in polyacrylamide gels. The ratio of –∂ (log D′)/∂T over KR increases with field strength in the range of 5–15 V/cm.
The determination of nitrite and nitrate in human blood plasma by capillary zone electrophoresis
Authors: Ueda Toshiko , Maekawa Tsuyoshi , Sadamitsu Daikai , Oshita Shuzo , Ogino Keiki , Nakamura Kazuyuki
Published: May 2005

The determination of nitrite and nitrate in human blood plasma by capillary zone electrophoresis (CZE) is described. Electrophoresis was carried out in a 65 cm long, 75 μm wide silica capillary, using 750 mM sodium chloride containing 5% NICE‐Pak OFM Anion‐BT (an additive reversing the electroosmotic flow) as running buffer, at a potential of 20 kV, with on‐column UV detection at 214 nm. Under these conditions, the two anions migrated in the order of nitrite and nitrate, completely resolved from each other. Forty‐one samples of ultrafiltered blood plasma of healthy volunteers were analyzed. The concentrations of nitrite and nitrate were 0.15 ± 0.07 mg/L and 3.2 ± 1.6 mg/L (mean± SD), respectively, with a linear correlation in the range of 0.1–50 mg/L. The within‐day and between‐day coefficients of variation (CVs) for nitrite and nitrate were lower than 5% and 8%, respectively. The recovery was 97‐114%. The estimated plasma volume needed for each determination was 108 nL.
Purification of wheat glutenin subunits by preparative acid and two‐dimensional electrophoresis
Authors: Curioni Andrea , Morel Marie‐Hélène , Furegon Luca , Redaelli Rita , Peruffo Angelo Dal Belin
Published: May 2005

Reduced alkylated glutenin subunits from wheat flour were fractionated by preparative electrophoresis at acid pH. The high molecular weight glutenin subunits (HMW‐GS) and some of the low molecular wight glutenin subunits (LMW‐GS) were purified by this one‐step procedure, whereas the remainder of the LMW‐GS, comigrating in the acid system, were purified in a second step by electroendosmotic preparative electrophoresis in the presence of sodium dodecyl sulfate. The quantities of recovered protein were sufficient for biochemical characterization and/or antibody production.
Serine protease inhibitor activity of recombinant squamous cell carcinoma antigen towards chymotrypsin, as demonstrated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis
Authors: Nawata Shugo , Tsunaga Nagato , Numa Fumitaka , Tanaka Tatehiko , Nakamura Kazuyuki , Kato Hiroshi
Published: May 2005

Squamous cell carcinoma (SCC) antigen was tested, by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, for its ability to inhibit the activity of serine proteases, i.e., trypsin, chymotrypsin and elastase. We demonstrated that the serine protease inhibitor (serpin) of SCC antigen is specific for chymotrypsin. Preincubation of chymotrypsin with recombinant SCC antigen inhibited chymotryptic digestion of gelatin and ovalbumin through the formation of sodium dodecyl sulfate‐stable complexes. These findings promote understanding of the biological functions of SCC antigen as serpin in the stratification of the normal squamous cells and in the malignant behaviour of the tumor cells.
Progress with gene‐product mapping of the Mollicutes: Mycoplasma genitalium
Authors: Wasinger Valerie C. , Cordwell Stuart J. , Cerpa‐Poljak Anne , Yan Jun X. , Gooley Andrew A. , Wilkins Marc R. , Duncan Mark W. , Harris Ray , Williams Keith L. , Humphery‐Smith Ian
Published: May 2005

A protein map of the smallest known self‐replicating organism, Mycoplasma genitalium (Class: Mollicutes), revealed a high proportion of acidic proteins. Amino acid composition was used to putatively identify, or provide unique parameters, for 50 gene products separated by two‐dimensional gel electrophoresis. A further 19 proteins were subjected to peptide‐mass fingerprinting using matrix‐assisted laser desorption ionisation‐time of flight (MALDI‐TOF) mass spectrometry and 4 were subjected to N‐terminal Edman degradation. The majority of M. genitalium proteins remain uncharacterised. However, the combined approach of amino acid analysis and peptide‐mass fingerprinting allowed gene products to be linked to homologous genes in a variety of organisms. This has allowed proteins to be identified prior to detection of their respective genes via the M. genitalium sequencing initiative. The principle of ‘hierarchical’ analysis for the mass screening of proteins and the analysis of microbial genomes via their protein complement or ‘proteome’ is detailed. Here, characterisation of gene products depends upon the quickest and most economical technologies being employed initially, so as to determine if a large number of proteins are already present in both homologous and heterologous species databases. Initial screening, which lends itself to automation and robotics, can then be followed by more time and cost intensive procedures, when necessary.
The major protein expression profile and two‐dimensional protein databse of human heart
Authors: Kovalyov Leonid I. , Shishkin Sergei S. , Efimochkin Andrei S. , Kovalyova Marina A. , Ershova Elisaveta S. , Egorov Tsezi A. , Musalyamov Alexsandr K.
Published: May 2005

The construction of a two‐dimensional protein database of the human heart is presented. The database contains information on about 300 abundant proteins of human myocardial tissue, including approximately 40 proteins that were identified by different methods. Each protein was characterized according to several parameters, including molecular weight, isoelectric point, name, partial sequence, subcellular localization, and genetic as well as embryonic changes.
Analysis of growth factor stimulated nucleocytoplasmic protein transport using two‐dimensional gel electrophoresis
Authors: Bedells Clare H. , Pennington Stephen R.
Published: May 2005

The rapid activation of gene expression by growth factors frequently involves the nucleocytoplasmic redistribution of proteins which regulate the transcription of discrete sets of genes. In particular, it has become apparent that protein kinases and transcription factors including protein kinase A, mitogen activated protein kinase, protein kinase C, NF‐χB and Stat91, can rapidly migrate from the cytosol to the nucleus in response to a wide variety of extracellular stimuli. Furthermore, it is assumed that these proteins represent important components of the signal transduction pathways which operate between the plasma membrane and the nucleus. Here two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) has been used to detect proteins which undergo rapid growth factor stimulated nucleocytoplasmic redistribution. Epidermal growth factor and insulin influenced the association of 15 individual protein spots with the nucleus. Estimates of the abundance of these proteins revealed that they represented approximately 1/1000 to 1/5000 of total nuclear protein. To permit identification by microsequencing, five of the protein spots have been recovered from multiple 2‐D PAGE gels and concentrated by one‐dimensional elution‐concentration gel electrophoresis. The data obtained demonstrate that 2‐D PAGE can be used to: (i) analyse putative regulators of growth factor activated gene transcription, and (ii) purify sufficient amounts of these proteins to facilitate identification by microsequence analysis.
New types of large‐pore polyacrylamide‐agarose mixed‐bed matrices for DNA electrophoresis: Pore size estimation from Ferguson plots of DNA fragments
Authors: Chiari Marcella , D'Alesio Luca , Consonni Roberto , Righetti Pier Giorgio
Published: May 2005

The average pore size value of gels containing polyacrylamide, covalently linked to agarose, was found to be 30% higher than the value of a regular N,N′‐methylenebisacrylamide (Bis) cross‐linked gel of the same %T. By increasing the agarose concentration (10% of the total amount of polyacrylamide), gels containing low amounts of acrylamide (1.5–2%) are reproducibly obtained; their pore sizes are 130% larger than the pore sizes of a 4%T, 3.3%C polyacrylamide gel. In general, mixed‐bed matrices were found to be more elastic and mechanically stronger than classical polyacrylamide gels since an agarose‐induced gelation process takes place during their polymerization.
Detection of biotinylated proteins in crossed immunoelectrophoresis gels: Studies on platelet membrane receptors and microparticles
Authors: Solum Nils Olav , Holme Pål André , Pedersen Turid M.
Published: May 2005

Biotinylation can be used as an alternative for surface labeling of cell membrane proteins. The use of the water soluble N‐hydroxysulfosuccinimide (NHSS)‐biotin or the more lipophilic N‐hydroxysuccinimide (NHS)‐biotin reagent has been investigated in the present study labeling two central receptor complexes on the platelet surface, i.e. the glycoprotein (GP) Ib–IX and the GP IIb–IIIa complexes involved in platelet adhesion and aggregation. Lack of labeling of the intracellularly located albumin was used as a negative control. The labeling has been studied using crossed immunoelectrophoresis in the PhastSystem format after extraction of the labeled cells in Triton X‐100, and it is shown that, using enzyme‐conjugated avidin and chromogenic substrates, the biotinylated proteins can be visualized directly in the dried electrophoresis gel without the need for a transfer to a blotting membrane as is used after sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Suitable conditions for biotinylation and for visualization in the crossed immunoelectrophoresis gels are described. Further, surface‐biotinylation of platelets was used to observe shedding of microparticles as a consequence of formation of the complement membrane attack complex. For this purpose the formation and composition of the biotinylated microparticles were observed by flow cytometry and crossed immunoelectrophoresis.
Capillary electrophoresis of carboxylated carbohydrates. Part 2. Selective precolumn derivatization of sialooligosaccharides derived from gangliosides with 7‐aminonaphthalene‐1,3‐disulfonic acid fluorescing tag
Authors: Mechref Yehia , Ostrander Gary K. , Rassi Ziad El
Published: May 2005

The most suitable conditions for selective precolumn derivatization of sialooligosaccharides, derived from gangliosides, with 7‐aminonaphthalene‐1,3‐disulfonic acid (ANDSA) and the subsequent separation of the derivatives by capillary electrophoresis are described. ANDSA‐sialooligosaccharide derivatives, which fluoresce at 420 nm when excited at 315 nm, were readily detected in capillary electrophoresis using an on‐column lamp‐operated fluorescence detector. In addition, the precolumn derivatization described here, which exploited the reactivity of the carboxylic acid group of the sialic acid residue of the oligosaccharides, replaced each weak carboxylic acid group of the parent sugar by two strong sulfonic acid groups. This allowed for electrophoresis over a wide range of pH and improved the resolution of the derivatives when compared to those obtained with underivatized sialooligosaccharides under identical separation conditions. The separation of sialooligosaccharides was best achieved when 75 mM borate, pH 10.0, was used as the running electrolyte. The derivatization and separation conditions described herein are expected to be readily transposed to the capillary electrophoresis of other sialooligosaccharides such as those derived from glycoproteins

Presented partially in a lecture (M‐L7) at the Fourth International Symposium Workshop on Bioanalysis, Lawrence, KS, July 10–13, 1994 and as part of a lecture at Electrophoresis Forum '94, Munich, Germany, October 24–26, 1994. Part I is reference [9].

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Separation of tryptophan‐derivative enantiomers with iron‐free human serum transferrin by capillary zone electrophoresis
Authors: Kilár Ference , Fanali Salvatore
Published: May 2005

Enantiomers can be separated by using human serum transferrin as a chiral phase. With the help of the native protein we were able to separate enantiomers with high efficiency, using a low ionic strength 2‐(N‐morpholino)ethanesulfonic acid (MES) buffer, pH 6, in capillary zone electrophoresis. Tryptophan methyl, ethyl and butyl ester enantiomers – moving towards the cathode at pH 6 – were resolved by passing through an iron‐free transferrin zone in coated capillaries. Since the isoelectric point of the iron‐free transferrin is a little higher than 6, the protein zone is either not moving in the experiment or is slowly moving towards the anode. Under the simplest experimental conditions the highest resolution was obtained for the butyl ester enantiomers and the lowest for the methyl ester ones. By changing the experimental conditions, however, this order could be reversed. The results indicate that the lengths of the alkyl chains in the enantiomers have a significant effect on the resolution, i.e., on the interaction between the protein and the separands.
Automated short tandem repeat (STR) analysis in forensic casework — a strategy for the future
Authors: Gill Peter , Kimpton Colin P. , Urquhart Andrew , Oldroyd Nicola , Millican Emma S. , Watson Stephanie K. , Downes Terry J.
Published: May 2005

Short tandem repeat (STR) loci are routinely analysed for forensic purposes in the UK. Because small regions of DNa are amplified, successful results are more likely to be obtained from highly degraded material where the DNA fragment length may be < 500 bp. The method is superceeding conventional analysis with single locus probes (SLPs). Dimeric STR loci display stutter artefacts, hence STRs used in casework are restricted to tri or tetrameric loci. Some STRs are complex repeats and have more alleles than simple repeats – for example the locus D21S11 has 21 alleles which differ in size by 2 bp because of the presence/absence of a hexanucleotide within the block of tetrameric repeats. These loci are of great potential interest because they combine increased discriminating power with reduced potential to stutter. Multiplexing 4 different loci with different dye labelled primers (i.e. carrying out polymerase chain reaction of 4 loci simulataneously) using the ABD 373 A automated sequencer enables a large numbers of samples to be processed. In addition data aquisition and manipulation is automated so that minimum postelectrophoresis operator input is required. It is our aim to develop a system equivalent in power to that of 4 single locus probes. To achieve this we have developed an octoplex system consisting of 7 loci and a sex test (amelogenin locus) which has a probability of chance of association of 10−9; the power of this system is equivalent to that achieved by 4 conventional SLPs.
Chemiluminescent detection of DNA probes in forensic analysis
Authors: Klevan Leonard , Horton Liz , Carlson David P. , Eisenberg Arthur J.
Published: May 2005

The conditions for hybridization and detection of enzyme‐labeled probes have been optimized in our laboratory for use with oligonucleotides coupled to alkaline phosphatase. We have examined several enzyme‐linked probes which are complimentary to commonly used variable number of tandem repeats (VNTR) loci to determine the feasibility of using chemiluminescence for routine application in forensic DNA analysis. It was found that a chemiluminescent detection system employing an alkaline phosphatase activated dioxetane in the presence of chemiluminescent enhancers provides a high degree of sensitivity in hybridization protocols with a significant savings in overall filter processing time. The chemiluminescent system achieved equal or greater sensitivity than observed for 32P‐labeled probes in much shorter development times. Furthermore, a new chemiluminescent substrate, Lumi‐Phos® Plus, has recently been investigated and found to further decrease the filter development time for forensic assays.
Simple protocols for typing forensic biological evidence: Chemiluminescent detection for human DNA quantitation and restriction fragment length polymorphism (RELP) analyses and manual typing of polymerase chain reaction (PCR) amplified polymorphisms
Authors: Budowle Bruce , Baechtel F. Samuel , Comey Catherine T. , Giusti Alan M. , Klevan Leonard
Published: May 2005

Methods for identity testing are described that enable extraction of DNA from biological samples, determination of the quantity of human DNA, and genetic analyses of the materials using restriction fragment length polymorphism (RFLP) typing and/or amplified fragment length polymorphism (AMP‐FLP) typing of PCR products. The salient features of the procedures are simplicity, manual typing, nonradioactive chemiluminescent assays or silver staining for detection, and low cost. Most application‐oriented laboratories involved in forensic and/or paternity testing should be able to implement these procedures.