bluuebunny/crossref_metadata_2025_split huggingface dataset:
A new multiphasic buffer system for sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of proteins and peptides with molecular masses 100 000–1000, and their detection with picomolar sensitivity
Authors: Wiltfang Jens , Arold Norbert , Neuhoff Volker 
Published: May 2005

AbstractA novel multiphasic buffer system for high resolution sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of dansylated and nondansylated proteins/peptides in the relative molecular mass (Mr) range of 100 000–1000 is described. The system, based on Jovin's theory of multiphasic zone electrophoresis [1–3], allows complete stacking and destacking of proteins/ peptides within the above Mr, range. The buffer system uses Bicine and sulfate as trailing and leading ion, respectively, and Bistris and Tris as counter ions in the stacking and separating phase, respectively. Through selection of two different counterions – the characteristic feature of the present ionic system – the stacking limits of a multiphasic buffer system can be further widened, thus making it applicable to gel electrophoresis of a larger spectrum of rapidly migrating species, such as sodium dodecyl sulfate‐proteins/peptides and nucleic acids, than has been possible previously. Highly sensitive detection methods for proteins as well as for polypeptides down to approximately Mr, 1000 are described. Dansylated proteins/peptides were detected by their fluorescence either directly within the gel or following electroblotting into anion‐exchange of polyvinylidene difluoride membranes. The latter procedure resulted in detection sensitivities of approximately 1 ng. Nondansylated proteins/peptides were either detected within the gel by colloi dal Coomassie staining or by electroblotting into polyvinylidene difluoride membranes, followed by colloidal gold staining. Prior to both staining procedures the proteins/peptides were pretreated with glutardialdehyde in the presence of borate at near neutral pH values to generate protein/peptide polymers of poor solubility. For a given pH the efficiency of the latter procedure was significantly influenced by the nature of the bufferion used in the fixation buffer. In contrast to conventional fixation procedures even small polypeptides (Mr 1000) were immobilized and approximately 15 ng and 0.75 ng could be detected after colloidal Coomassie and colloidal gold staining, respectively.
High‐performance capillary electrophoresis of gangliosides
Authors: Liu Yuanbin , Chan K.‐F. Jesse 
Published: May 2005

AbstractGangliosides are sialic acid‐containing glycosphingolipids. In aqueous media, these glycolipids have been shown to exist as stable micelles. Ganglioside micelles could be analyzed by high‐performance zonal capillary electrophoresis in uncoated fused‐silica capillaries within 10 min. The mass sensitivity determined by monitoring the absorption of ultraviolet light at 195 nm was in the order of 10−11 mol. Increasing the pH of the running buffer from 3.0 to 7.4 or the voltage from 10 to 30 kV increased the relative mobilities of gangliosides. By contrast, increasing the ionic strength of the buffer decreased the migration and broadened the elution peak widths of gangliosides. GangliosideThe abbreviations used for gangliosides are according to the recommendations of the Nomenclature Committee, International Union of Pure and Applied Chemistry [46]. The basic designation of ganglio‐series gangliosides are based on the Svennerholm system [47]. For example: GMl, II3NeuAc‐GgOse4Cer.
micelles including GM1, GD1b and GT1b were resolved into separate peaks by capillary electrophoresis at physiological pH shortly after mixing. Upon prolonged inc ubation, the ganglioside peaks merged to form a single species. The fusion process was temperature‐dependent. At 50°C, formation of mixed micelles between polysialogangliosides GD1b and GT1b was complete within 30 min. In contrast, no fusion of the ganglioside peaks was observed at 0°C even after 75h. Formation of mixed micelles between GD1b and other polysialogangliosides including GD1a, GT1b and GQ1b at 37°C required 1.5, 3.0 and 2.0 h, respectively. Formation of mixed micelles between monosialoganglioside GM1 and polysialogangliosides were 6‐ to 36‐fold slower. No fusion was observed between monosialogangliosides GM1 and GM2 after 2 days of incubation. These findings indicate that polysialogangliosides may have higher propensities than monosialoganglioside to form mixed micelles.
Quantitative analysis of two‐dimensional protein profiles of inbred lines of maize (Zea mays L.)
Authors: Higginbotham Jeri Welsh , Smith J. Stephen C. , Smith Oscar S. 
Published: May 2005

AbstractTwo‐dimensional electrophoresis and fluorography of [35S] methionine labeled maize germinated embryo proteins were performed at Cold Spring Harbor Laboratory. Fluorographs of 63 gels representing 37 inbred lines were subsequently scanned and spot‐detected at Protein and DNA Imageware Systems (Huntington Station, NY). The digitized images were then matched with the aid of PDQUESTII computer software. Over 1500 different protein spots were included in the resulting dataset. The optical density data were normalized to parts per million, then transformed to their natural logarithms. Analyses of variance were performed on each spot in order to select for further study those spots with most of their variation partitioned among inbred lines rather than within inbred lines. Using this method of spot selection, over 100 protein spots were included in the set of spots which display significant differences among inbred lines of maize.
The simplified technique of high resolution two‐dimensional polyacrylamide gel electrophoresis: Biomedical applications in health and disease
Authors: Marshall Thomas , Williams Katherine M. 
Published: May 2005

AbstractThe application of our simplified technique of high resolution two‐dimenional polyacrylamide gel electrophoresis (2‐D PAGE) to human body fluids is reviewed. Serum/plasma protein changes associated with alcohol abuse, familial dyslipoproteinemia (“fish‐eye” disease), and myocardial infarction are demonstrated. High resolution 2‐D PAGE of amniotic fluid, cerebrospinal fluid, urine, and saliva is shown with reference to the work of others, and the detection of pink‐violet staining “lumicarmines” in sweat and tear fluid is reported for the first time. General aspects relating to the methodology are discussed. These include sample preparation, the choice of electrophoresis conditions (denaturing or nondenaturing) and detection method (Coomassie Brilliant Blue or silver), and the effects of native protein pretreatment with sodium dodecyl sulfate prior to silver staining or isoelectric focusing gel shrinkage in glycerol prior to second‐dimensional sodium dodecyl sulfate‐polyacrylamide gel electrophoresis.
Use of a fast protein electrophoretic purification procedure for N‐terminal sequence analysis to identify S‐locus related proteins in stigmas of Brassica oleracea
Authors: Gaude Thierry , Denoroy Luc , Dumas Christian 
Published: May 2005

AbstractIn the cruciferous plant Brassica oleracea L. (cabbage), the S‐locus specific glycoproteins (SLSGs) isolated only in stigmas are considered to play an important role in the normal prevention of self‐fertilization. Recent molecular data have shown that the gene encoding these glycoproteins (the SLG gene) belonged to a multigenic family consisting of about 10 homologous copies among which an, other member is expressed, the S‐locus related gene (SLR1gene). Our aim was to determine whether the SLR1‐gene proteins were expressed in the stigmatic tissues. We first identified the putative SLSGs or SLR1‐proteins by Con A‐peroxidase detection of glycoproteins separated after isoelectric focusing in polyacrylamide gels. We describe a fast purification procedure for the glycoproteins of interest, based on analytical isoelectric focusing, electrophoresis, and electroblotting of proteins onto polyvinylidene difluoride membranes. Blotted proteins were sequenced for N‐terminal amino acid determination. By comparison of the N‐terminal sequences of the purified proteins with the peptide sequence predicted from the SLR1‐cDNA, we demonstrate the expression of SLR1‐like proteins in stigmas of B. oleracea.
Genetic polymorphisms of the A and B subunits of human coagulation factor XIII in mainland Italy and Sardinia: Description of a new FXIIIA variant allele
Authors: Scacchi Renato , Corbo Rosa Maria , Mulas Giuseppe , Mureddu Libero , Pascone Roberto 
Published: May 2005

AbstractThe distribution of the two alleles of FXIIIA and the three alleles of FXIIIB were studied in populations from mainland Italy and from Sardinia. The frequencies of the FXIIIA*2 allele were 0.266 and 0.265. The frequencies of FXIIIB*1 were 0.787 and 0.765; of B*2, 0.070 and 0.094; of B*3, 0.143 and 0.141. A new cathodal FXIIIA allele (A*7) was described in the Rome sample. No significant difference in the distribution of allele frequencies for either system was found between the two populations studied. For typing both markers, good results were also obtained by using whole blood instead of plasma.
The characterization and use of different antibodies against the hsp70 major heat shock protein family for the development of an immunoassay
Authors: Margulis Boris A. , Nacharov Peter V. , Tsvetkova Olga I. , Welsh Michael , Kinev Alexander V. 
Published: May 2005

AbstractThe hsp70 family of major stress proteins is composed of several different members exhibiting similar structural and functional properties. In order to obtain an antiserum with wide epitope reactivity, rabbits were immunized with a mixture of native and denatured hsp70 purified from bovine muscle by ATP‐affinity chromatography. Screening for antibody specificity was performed by a “sandwich” enzyme linked immunosorbent assay (ELISA). Immunoprecipitation and immunoblotting analyses demonstrated that the polyclonal antiserum obtained by us and a monoclonal antibody raised against a different preparation of antigen recognized the same determinant on the native hsp70 molecule (inducible form). With a different specificity the polyclonal antiserum recognized only the denatured monomers of the other members of the hsp70 family. These results are discussed in relation to the immunological features of the hsp70 molecule and to the development of an immunoassay for the detection of hsp70 in Cell and tissue extracts.
Improved method for identification of proteins using two‐dimensional electrophoresis with immobilized pH gradient isoelectric focusing
Authors: Mackin Robert B. , Raker Veronica A. , Spiess Joachim 
Published: May 2005

AbstractRecent advances in protein sequence analysis now permit the determination of partial N‐terminal and internal primary structure from low picomole quantities of protein. The major remaining hurdles to sequence analysis of small amounts of protein are the identification, isolation, and handling of microgram and submicrogram quantities of protein. The technique of two‐dimensional Electrophoresis using immobilized pH gradient isoelectric focusing circumvents many of these problems. However, poor correlation between the first and second dimension have prevented use of this technique for the identification of some proteins which can only be assayed prior to the denaturing conditions used in the second dimensional sodium dodecyl sulfate‐polyacrylamide gel Electrophoresis procedure. An improved method is presented which allows correlation of the native biological activity (first dimension) to a silver stained protein (second dimension) with a high degree of confidence.
The MELANIE project: From a biopsy to automatic protein map interpretation by computer
Authors: Appel Ron D. , Hochstrasser Denis F. , Funk Matthieu , Vargas J. Reynaldo , Pellegrini Christian , Muller Alex F. , Scherrer Jean‐Raoul 
Published: May 2005

AbstractThe goals of the MELANIE project are to determine if disease‐associated patterns can be detected in high resolution two‐dimensional polyacrylamide gel electrophoresis (HR 2D‐PAGE) images and if a diagnosis can be established automatically by computer. The ELSIE/MELANIE system is a set of computer programs which automatically detect, quantify, and compare protein spots shown on HR 2D‐PAGE images. Classification programs help the physician to find disease‐associated patterns from a given set of two‐dimensional gel electrophoresis images and to form diagnostic rules. Prototype expert systems that use these rules to establish a diagnosis from new HR 2D‐PAGE images have been developed. They successfully diagnosed cirrhosis of the liver and were able to distinguish a variety of cancer types from biopsies known to be cancerous.
A two‐dimensional electrophoresis‐related laboratory information processing system: Spot matching
Authors: Kuick Rork D. , Skolnick Michael M. , Hanash Samir M. , Neel James V. 
Published: May 2005

AbstractAn approach for the computer‐assisted analysis of two‐dimensional gels has been developed as a part of our laboratory information processing system (LIPS). This approach relies in part on an algorithm for the pairwise matching of protein spots. The matching process initially matches spots based on a cross‐correlational measure of how well neighboring spots align. While this first pass correctly determines most spot correspondences and noncorrespondences, it can make errors. Higher accuracy is obtained by monitoring the consistency of spot match decisions in a second pass, which demands that neighboring spot pairs that align spatially must also have been found to match in the first pass. Pairwise comparisons of gels are, combined into n‐way comparisons by matching spot lists of gels to “master” gel spot lists, which in turn are matched to higher level masters, resulting in a hierarchy of matched spots. After each pairwise match the results are reviewed and corrected with the assistance of a graphical match‐editor. Results are given for 19, single‐Cell‐derived lymphoid clones in which the presence of a mutation had previously been established, each processed in duplicate. Only one of 46 spot changes failed to be detected, which demonstrates that the strategy is sensitive and efficient for detecting qualitative spot differences.
Immobilized pH gradients (IPG) simulator ‐ an additional step in pH gradient engineering: II. Nonlinear pH gradients
Authors: Righetti Pier Giorgio , Tonani Carlo 
Published: May 2005

AbstractWhile in the companion paper (Tonani, C. & Righetti, P. G., Electrophoresis 1991, 12, 1011–1021) we gave the general outline of our new computer program, immobilized pH gradients (IPG) simulator, able to simulate and optimize linear pH gradients for isoelectric focusing in immobilized pH gradients, in the present report we extend the application of such a program to: (i) convex exponential gradients, (ii) logarithmic and (iii) polynomial gradientsProgram available from Fluka Chemie AG, Buchs, Switzerland
. Such gradients are meant to give equal space to protein spots in complex protein mixtures (e.g., cell lysates, biological fluids) and follow the statistical distribution of protein pI values along the pH axis. They will prove of fundamental importance in two‐dimensional maps, both because they optimize the spreading of spots in the two‐dimensional plane and because of the excellent reproducibility of immobilized pH gradients. The following concave exponential recipes are given: pH 3–8, pH 3–9, pH 3–10, pH 3–11, pH 4–7, pH 4–8, pH 4–9, pH 4–10, pH 4–11, pH 5–8, pH 5–9, and pH 5–10, as well as the most extended pH 2.5–11 interval. Two interesting logarithmic gradients are described: pH 3–6 and pH 3–7 and one sigmoidal (derived with a polynomial of 5th degree): pH 3–11.
Capillary electrophoresis of DNA in agarose solutions at 40°C
Authors: Boček Petr , Chrambach Andreas 
Published: May 2005

AbstractDNA fragments ranging from 72 to 1353 by in length (ϕX174 RF DNA/HaeIII) were separated by capillary electrophoresis in 0.3–2.0% solutions of agarose (SeaPlaque GTG) at 40°C. Liquified agarose above its gelling temperature is easily filled and refilled into capillaries. Its background absorbance at 260 nm was sufficiently low to allow for DNA detection at an estimated DNA load of 13 ng/10 components. Sample injection proceeded at 1 kV for 16s. The internal capillary diameter was 150 μ, the migration path 27 cm. Migration times varied from 5 to 14 min at 185 V/cm. Potentially, the applicability of capillary electrophoresis in agarose solutions can be expected to extend to the entire size range of DNA, in view of the recent demonstration of kb‐sized circular DNA separations in agarose solutions, and those of Mb‐sized DNA‐agarose complexes in linear polyacrylamide solutions.
Capillary electrophoresis in agarose solutions: Extension of size separations to DNA of 12 kb in length
Authors: Boček Petr , Chrambach Andreas 
Published: May 2005

AbstractThe upper limit of the size range of DNA amenable to separation in agarose solutions above their gelling temperature, using capillary zone electrophoresis apparatus, was increased to 12 kb. The plot of log(bp)vs. mobility derived from electrophoresis in 1.7% agarose solution is biphasic, exhibiting higher resolving power for DNA less than 1 kb in size than that of larger sizes. Resolving power for DNA larger than 1 kb increased when the agarose concentration was increased in the range of 1.0–2.6%. It was similar in solutions at 40°C of SeaPrep and SeaPlaque agaroses as well as in Acrylaide (trade names are those of the manufacturer). However, the resolving power of SeaPrep agarose at 25°C was inferior to that at 40°C. Concave plots of log(mobility) vs. concentration of the agarose solutions are those predicted under the assumption that the effective “equivalent radius” of the DNA molecule diminishes with increasing agarose concentration in the investigated concentration range up to 2.6%.
Continuous, on‐line DNA sequencing using a versatile infrared laser scanner/electrophoresis apparatus
Authors: Middendorf Lyle R. , Bruce John C. , Bruce Robert C. , Eckles Robert D. , Grone Daniel L. , Roemer Stephen C. , Sloniker Gary D. , Steffens David L. , Sutter Scott L. , Brumbaugh John A. , Patonay Gabor 
Published: May 2005

AbstractA new apparatus for continuously detecting fluorescently labeled DNA fragments is based on infrared fluorescence technology. This technology combines state‐of‐the‐art developments in chemistry, laser technology, and detection, while achieving improved reliability, sensitivity, and flexibility for applications including DNA sequencing. DNA molecules labeled with a novel infrared fluorophore are detected during electrophoresis using a scanning infrared fluorescence microscope. The microscope consists of a laser diode for exciting the fluorophore and a silicon avalanche photodiode for detecting the infrared emission. Optimum conditions for detection and throughput are obtained by adjusting electrophoresis, scanning and imaging parameters. Typical DNA sequencing runs (test templates) allow identification of over 500 bases per sample with > 99% accuracy.
High yield electroblotting onto polyvinylidene difluoride membranes from polyacrylamide gels
Authors: Mozdzanowski Jacek , Hembach Peter , Speicher David W. 
Published: May 2005

AbstractOptimal conditions of electroblotting that led to high protein recovery on polyvinylidene difluoride (PVDF) membranes were determined for sodium dodecylsulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). SDS concentrations in the gel and transfer buffer were found to be the most important factors affecting the amount of protein recovered on the PVDF membrane. The largest loss occurred during the first 10–30 min of transfer due to the relatively high initial SDS concentration in the gel. During this initial stage of transfer, most of the protein passed through the primary membrane and was partially retained on secondary and tertiary membranes. The value of presoaking gels prior to transfer to reduce the amount of SDS was evaluated by quantitating free SDS densitometrically and by correlating the reduced SDS concentration with increased electroblotting efficiency from presoaked gels. Transfer time was evaluated and no “overtransfer” was found even after very long transfer times. These results clearly indicate that proteins electroblotted onto PVDF membranes were tightly bound and could not be released by extending the transfer time. The effects of methanol and SDS concentrations on protein adsorption from solution to PVDF were also determined quantitatively. The results of this study strongly suggest that proteins fully saturated with SDS cannot bind efficiently to PVDF membranes. Since SDS is necessary for high protein mobility, the challenge in efficient electroblotting is to maintain an optimal SDS concentration which is high enough to permit effective removal from the gel and low enough to permit effective binding to the PVDF membrane. For 1.5 mm thick gels containing 0.2% SDS, presoaking the gel for 15–20 min in transfer buffer with 10% methanol prior to electroblotting provided the best recovery on the primary membrane.
Temperature‐gradient gel electrophoresis for the detection of polymorphic DNA and for quantitative polymerase chain reaction
Authors: Riesner Detlev , Steger Gerhard , Wiese Ulrich , Wulfert Michael , Heibey Monika , Henco Karsten 
Published: May 2005

AbstractIn order to detect mutations in a gene, either known mutations from human diseases or artifical ones in transgenic animals, or to screen for not yet identified mutations in patients, a method is required which guarentees detection of mutations which might occur in every single position of the whole open reading frame (ORF). It will be shown that a combination of polymerase chain reaction (PCR) and temperature gradient gel electrophoresis (TOGE) fulfills these requirements. By thermodynamic calculations the shift in the gel electrophoresis due to a mutation can be calculated in dependence on the position of the mutation. The theoretical results were tested with the mutations known so far. The quantitative determination of the copy number of a specific DNA or RNA sequence in a biological specimen (quantitative PCR) can be performed precisely and easily by combining PCR and TGGE. The system uses a quantification strategy of a new type of internal standardization. TGGE is applied to separate homo‐ and heteroduplexes which correspond respectively to standard and template sequences. The accuracy of this quantification strategy is very high, with a variability of <15 %. In addition to quantification, PCR/TGGE detects PCR artifacts and template mutants.
Nonradioactive ribonuclease protection analysis using digoxygenine labeling and chemiluminescent detection
Authors: Wundrack Iris , Dooley Steven 
Published: May 2005

AbstractA sensitive nonradioactive ribunouclease protection assay is described which we have used to study c‐myc gene transcription and promoter usage in GLC4, a human small cell lung carcinoma cell line with amplified gene. For in vitro transcription we used digoxygenine (DIG)‐rUTP instead of [α‐32P]CTP or [α‐32P]UTP and after polyacrylamide gel electrophoresis the protected probes were transferred to a nylon membrane from Boehringer Mannheim using electroblotting. Subsequently the membrane was analyzed by chemiluminescent detection. Results were obtained after 1 h of exposure and were comparable with those using radioactivity.