bluuebunny/crossref_metadata_2025_split huggingface dataset:
Sample streaks and smears in immobilized pH gradient gels
Authors: Esteve‐Romero Josep , Simò‐Alfonso Ernesto , Bossi Alessandra , Bresciani Filippo , Righetti Pier Giorgio 
Published: May 2005

AbstractIn immobilized pH gradient (IPG) gel formulations as wide as PH 4–9, encompassing neutrality and containing the pK 7.0 acrylamido buffer as one of the buffering ions, smears are directly proportional to the total amount of the pK 7.0 species. At a total level of 10 mM pK 7.0 in these gel formulations, severe smears occur not only for mildly hydrophobic proteins (e.g., recombinant alcalase and termamylase) but also for the relatively hydrophilic pI marker proteins. Streaks and smears are essentially abolished in recipes devoid of the pK 7.0 compound or in formulations containing a maximum of 3 mM of this component. Although partitioning in water/n‐octanol has shown the pK 7.0 acrylamido buffer to be quite hydrophobic (P = 0.5), the occurrence of smears could be to the presence of oligomers in some commercial preparations. Even when dissolved in n‐propanol, some batches of acrylamido buffers might still contain oligomers, probably formed during the synthetic step.
Novel acrylamido monomers with higher hydrophilicity and improved hydrolytic stability: I. Synthetic route and product characterization
Authors: Simò‐Alfonso Ernesto , Gelfi Cecilia , Sebastiano Roberto , Citterio Attilio , Righetti Pier Giorgio 
Published: May 2005

AbstractThe novel acrylamido monomer reported by our group (N‐acryloylaminoethoxyethanol, AAEE; Chiari et al., Electrophoresis 1994, 15, 177–186), found to combine high hydrophilicity with extraordinary resistance to alkaline hydrolysis, has come under closer scrutiny due to unexpected and random autopolymerization while stored as a 1/1 v/v water solution at 4°C (possibly due to a greater oxidability of the ether group). We have additionally found a unique degradation pathway of the monomer, called “1–6 H‐transfer”, by which the C1 (on the double bond site), by constantly ramming against the C6, next to the ether oxygen (O7, which in fact favors the transfer of the hydrogen atom by C1), produces radicals which more efficiently add to the monomer favoring autopolymerization and cross‐linking. A number of novel monomers is proposed while maintaining the other unique chracteristics of AAEE. One of them, N‐acryloylaminopropanol, offers all the unique, special qualities of AAEE, without the noxious aspects of autopolymerization. Additionally, a synthetic route was optimized, yielding an essentially pure product in a single reaction step, with a yield >99% and an equivalent purity (>99%). The synthesis consists in reacting acryloyl chloride at −40°C in presence of a twofold molar excess of aminopropanol and in ethanol (instead of methanol) as solvent. Other solvents, as well as the use of triethylamine for neutralizing the HCl produced, were found to give a variety of undesired byproducts.
Isolation of proteins and nucleic acids by electrophoresis on disposable gel columns
Authors: Costa Maria A. , Duro Giovanni , Porcasi Rossana , Colombo Paolo , Locorotondo Giovanni , Geraci Domenico , Izzo Vincenzo 
Published: May 2005

AbstractA simple and cheap one‐step method to isolate proteins or nucleic acids by electrophoresis in disposable gel columns is reported. A disposable syringe was modified to host a gel column and an elution chamber. Starting from a crude extract of E. coli, the laboratory‐made devise allowed the isolation of the maltose binding protein (MBP) fused to a recombinant allergenic molecule with a molecular mass of 58 kDa, from a mixture of several proteins. Also, plasmid DNA could be isolated from a mixture containing chromosomal DNA and RNA, avoiding the use of organic solvents. Electrophoresis was performed at 150 V, 35°C, pH 8.0 and 8.3 for protein and DNA, respectively. The protein or the DNA obtained showed a yield of 80% and a purity grade of 90%, as estimated by densitometry.
Identification of stress proteins in lysates of human cell lines separated by two‐dimensional electrophoresis and electroblotted simulataneously onto two different membranes
Authors: Vogt Christian P. , Willi Annemarie , Hess Daniel , Hunziker Peter E. 
Published: May 2005

AbstractA protocol based on a combination of established methods for the characterization and identification of inducible stress proteins in human cell lines is described. A particular protein spot, collected from several micropreparative two‐dimensional electrophoresis (2‐DE) gels, is concentrated into a new gel prior to simultaneous electrotransfer onto a Cationic Durapore (CD) membrane and onto a polyvinylidene (PVDF) backup membrane. The protein blotted onto the PVDF support is subjected to N‐terminal sequence analysis. From the protein bound to the CD membrane the peptide mass profile is obtained by proteolytic digestion of the protein followed by the separation of the resulting peptides by high performance liquid chromatography (HPLC) and their detection by on‐line electrospray mass spectrometry (LC/MS). Additional internal sequence information may be obtained by amino acid sequence analysis of peptides collected in the HPLC effluent. The efficiency of this strategy is demonstrated with two proteins extracted from 15 micropreparative 2‐DE gels of an extract of a human liver cell line. The peptide mass fingerprinting of a 60 kDa protein with a pI of 5.3 assigned 22 of 37 peptides to the heat shock protein 60 (Hsp 60). The result was confirmed by the N‐terminal sequence analysis of the undigested protein and of an internal tryptic fragment. The second sample, a 40 kDa protein with a pI of 4.9, was identified as a processed form of the heat shock cognate 71 kDa protein (Hsc 70).
Sequence patterns produced by incomplete enzymatic digestion or one‐step Edman degradation of peptide mixtures as probes for protein database searches
Authors: Jensen Ole N. , Vorm Ole , Mann Matthias 
Published: May 2005

AbstractMass spectrometric peptide mapping of proteins isolated by polyacrylamide gel electrophoresis is a rapid method for identifying proteins in sequence databases. A majority of tryptic peptide maps were found to contain pairs of peptide ion peaks separated by the molecular weight of the lysyl or arginyl residue. These peaks originate from amino acid sequence patterns such as Lys‐Lys where trypsin has cleaved C‐terminals to either one of the lysines. The peptide mass and the pattern define an N‐ or C‐terminal sequence tag. Searching sequence databases by such a sequence tag results in only a moderate number of matches and significantly reduces the number of database matches when used in combination with a peptide mass map. Two N‐ or C‐terminal sequence tags alone unambiguously identify a protein in most cases. The technique discussed here is simple, does not require additional measurments, and increases the percentage of protein samples that can be identified by their mass maps alone. N‐Terminal peptide sequence tags for database searching can also be generated by manual one‐step Edman degradation of the unseparated peptide mixture.
Real‐time velocity of DNA bands during field‐inversion gel electrophoresis
Authors: Sabanayagam Chandran R. , Holzwarth George 
Published: May 2005

AbstractThe velocity v of bands of double‐stranded, linear DNAs containing 48.5–5700 kbp was determined with 0.3 s resolution during field‐inversion agarose gel electrophoresis (FIGE) for a broad range of the forward pulse period T+, keeping the duration of the backward pulse T− = T+/3. Within 0.6 s or less after the field changed sign from − to +, the velocity showed a sharp positive peak; a similar spike, but with negative velocity, occurred immediately after the field changed from + to −. For long pulses, the magnitude of this spike increased with M0.36, reaching ten times the steady‐state velocity for M = 5.7 kbp. After this spike, the velocity dipped to 55–75% of its value in a steady field, then increased to a small secondary peak before reaching a steady‐state plateau. The duration of the velocity trough, and the time of the small peak, increased as M1. For standard FIGE conditions (ratio of forward:reverse pulse duration, T+:T− = 3:1; equal forward and reverse field amplitudes, E+ = E−), the mobility μ = ∫ vdt over a complete cycle was a minimum when E+ terminated at the end of the velocity trough. The minimum occurred because the velocity during E+ sampled primarily the trough, and because the backward velocity during E− was exceptionally large; the negative velocity spike was maximized when T+ terminated at the end of the velocity trough. Computer simulations of FIGE by Zimm (J. Chem. Phys. 1991, 94, 2187–2206) and by Duke and Viovy (J. Chem. Phys. 1992, 96, 8552–8563) generate real‐time velocities that are in excellent agreement with our experimental data.
Conformational dynamics of DNA during biased sinusoidal field gel electrophoresis
Authors: Masubuchi Yuichi , Oana Hidehiro , Matsumoto Mitsuhiro , Doi Masao , Yoshikawa Kenichi 
Published: May 2005

AbstractDNA motion during biased sinusoidal field gel electrophoresis around the antiresonance condition was investigated by direct observation using fluorescent microscopy and Brownian dynamics simulation. Time development of the center of mass velocity, vx, and the principal value of the gyration tensor, Rl, was measured at this antiresonance condition. The typical stretch‐contract motion, which is observed in steady field and at high frequency field, is severely suppressed, and there are two or more dominant kinks that compete with each other. Analysis of the kink motion from the simulation results supports this picture.
Fluorescence resonance energy transfer visualization of DNA aggregates formed during electrophoretic separations in ultradilute hydroxyethylcellulose
Authors: Schwinefus Jeffrey James , Wang Shau‐Chun , Hammond Richard W. , Morris Michael D. 
Published: May 2005

AbstractFluoresence resonance energy transfer (FRET) microscopy is used to observe structures of DNA aggregates formed during electrophoresis in ultradilute hydroxyethylcellulose (HEC). FRET gives evidence of aggregates with a tightly packed core and random entanglement of DNA in the outer portions. HEC concentrations also appear to play a role in aggregate formation. DNA aggregates take longer to assemble in higher HEC concentration solutions and assume a less compact form than those formed at lower HEC concentrations.
Protein separation by electrophoresis in a nonsieving amphoteric medium
Authors: Blanco Stéphane , Clifton Michael J. , Joly Jean‐Louis , Peltre Gabriel 
Published: May 2005

AbstractA numerical model has been used to study the separation of protein mixtures by zone electrophoresis in a nonsieving amphoteric medium. An amphoteric buffer fixes the pH of the solution close to its isoelectric point, where the buffer molecules are uncharged: they thus contribute very little to the conductivity of the solution. This means that high field strengths can be used for rapid separation without sacrificing resolution. The numerical study shows that in this process the band spreading that can reduce resolution is essentially due to differences in migration rate between protein‐rich and low‐protein zones: both differences in solution conductivity and in protein mobility are involved. Rules for judging the buffer capacity of amphoteric molecules are presented and it is shown how, for a given protein, the effectiveness of this technique varies with the range of pH in which it is applied.
Purification of thermamylase in multicompartment electrolyzers with isoelectric membranes: The problem of protein solubility
Authors: Esteve‐Romero Josep S. , Bossi Alessandra , Righetti Pier Giorgio 
Published: May 2005

AbstractThe main α‐amylase from Bacillus licheniformis (called thermamylase because of its resistance to high temperatures, 90°C) has been subjected to purification by isoelectric focusing in multicompartment electrolyzers with isoelectric membranes. The enzyme tended to precipitate, producing severe smears in proximity of its pI value (7.18). Solubility could not be ameliorated by any of the known means typically adopted in isoelectric focusing and compatible with enzyme activity, such as addition of neutral and zwitterionic surfactants (e.g., Nonidet, 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propane‐sulfonate, up to 2%), mixed hydro‐organic solvents (glycerol, ethylene glycol, propylene glycol) and addition of zwitterions unable to form micelles, such as taurine. However, addition of sugars, notably saccharose, sorbitol, and, to a lesser extent, sorbose, greatly improved protein solubility in the pI proximity. The improvement was dramatic if these sugars were admixed with 0.2 M taurine. Additionally, the increment of solubility (which occurred when reaching a level of 40% of the different sugars) was accompanied by a large pI shift, typically reducing the pI value by as much as 0.4 pH units (e.g., from a pI of 7.18 in the absence of additives to a pI of 6.80 in presence of a mixture of 40% sucrose and 0.2 M taurine, the best solubilizer in all the series investigated). This apparent pI shift was not due to a change of pH gradient caused by the presence of additives, since pH measurements in the absence as well as presence of additives gave identical results. The results are explained by the theory of Timasheff and Arakawa on stabilization of protein structure by solvents: sugars (at ca. 1 M concentration) and zwitterions such as taurine belong to class I stabilizers, characterized by negative binding to proteins and by increasing the surface tension of water. As a result, the protein is in a state of “superhydration”, which might prevent binding to Immobilines in the gel matrix and might alter some pKs on the protein surface. In solutions of 40% saccharose and 0.2 M taurine, thermamylase could be successfully purified to a single isoelectric and isoionic band in the multicompartment electrolyzer.
Validation of highly discriminating multiplex short tandem repeat amplification systems for individual identification
Authors: Kimpton Colin P. , Oldroyd Nicola J. , Watson Stephanie K. , Frazier Rachael R. E. , Johnson Peter E. , Millican Emma S. , Urquhart Andrew , Sparkes Becky L. , Gill Peter 
Published: May 2005

AbstractShort tandem repeat (STR) loci are routinely employed for individual identification. We have examined the performance and reproductibility of a highly informative co‐amplification system containing the tetranucleotide STR loci: HUMVWFA31/A, HUMTH01, D20S85, D8S1179, HUMFIBRA, D21S11, and D18S51, in conjunction with the amelogenin sex test, in addition to a modified system omitting the locus D20S85. Polymerase chain reaction (PCR) products were fluorescently detected on an automated sequencer and automatically sized against an internal size standard by Genescan software. Both systems were routinely able to type 500 pg of undegraded DNA. At DNA concentrations between 50–500 pg, partial profiles were produced, but no allelic drop‐out was observed. Balanced amplification of all loci occurred over a wide range of DNA concentrations from 50 pg to 10 ng. Alteration of reagent concentrations and cycling parameters from optimal resulted in variation in the efficiency of individual locus amplification relative to the other loci within the system. This was also observed at high ionic strength or extreme pH. However, at all reagent concentrations and conditions, allelic drop‐out was not observed. These multiplex systems have potential in both routine forensic and intelligence database applications.
Comparison of different electrophoretic methods for digital typing of the MS32 (D1S8) locus
Authors: Rodriguez‐Calvo María Sol , Bellas Susana , Soto Jose Luis , Barros Francisco , Carracedo Angel 
Published: May 2005

AbstractMinisatellite variant repeat mapping by polymerase chain reaction (MVR‐PCR) is a new and powerful approach for individual identification from human DNA. This method has been successfully applied to the hypervariable human minisatellite MS32 (locus D1S8). In this work, we further investigate this locus using different electrophoretic techniques which include: agarose gels and nonradioactive hybridization, miniaturized polyacrylamide gels, followed by silver staining, and automatic detection of fluorescent labeled alleles. The advantages and disadvantages of each method are described. Also, MVR codes from a Galician population (NW Spain) are provided.
An exactly solvable Ogston model of gel electrophoresis II. Sieving through periodic gels
Authors: Slater Gary W. , Guo Hong L. 
Published: May 2005

AbstractRecently, we developed a lattice model to study the dynamics of particles being electrophoresed in gels (G. W. Slater, H. L. Guo, Electrophoresis 1995, 16, 11–15). In Part I of this series (G. W. Slater, H. L. Guo, Electrophoresis 1996, 17, 977–988), we showed how to calculate the exact electrophoretic mobility of one‐site particles in the limit where the electric field intensity E is vanishingly small. Since we can solve the model for arbitrary gel structures in two or more dimensions, we compared our results with those of the Ogston‐Morris‐Rodbard‐Chrambach model (OMRCM) of gel electrophoresis, which assumes that the mobility (μ) of charged particles is directly proportional to the fractional gel volume (f) that is available to them. Our results and theoretical analysis indicated that the OMRCM is a mean‐field approximation that can be useful as a rough guide; however, it generally misses the subtle sieving effects related to the correlations between the position of the obstacles in a given gel structure. In this paper (Part II) we study, for two‐dimensional periodic gels, the exact relationships between the zero‐field mobility μ and the gel concentration C for larger particle sizes. The fact that μ is a strong function of the particle size suggests that we can separate large particles using two‐dimensional periodic gels (similar to those fabricated by W. D. Volkmuth and R. H. Austin, Nature 1992, 358, 600–602). We analyze our data using Ferguson‐like plots and we show that one can indeed use a generalized retardation coefficient, K, to estimate the effective pore size, aK and effective fiber size rK for these model gels. We conclude that the retardation coefficient is a useful concept to characterize a sieving structure even though it does not permit the inference of the exact gel structure.
Electrophoresis of DNA in novel thermoreversible matrices
Authors: Sassi Alexander P. , Barron Annelise , Alonso‐Amigo M. Goretty , Hion Daniel Y. , Yu Jessina S. , Soane David S. , Hooper Herbert H. 
Published: May 2005

AbstractWe demonstrate the feasibility of temperature‐sensitive polymers as novel matrices for capillary and slab electrophoresis of DNA. These matrices combine the ease‐of‐use of agarose with resolution properties of polyacrylamide. Two classes of matrices are used: (i) aqueous suspensions of gel microspheres and (ii) solutions containing uncross‐linked temperature‐sensitive polymers. When heated, the viscosity of these solutions drops dramatically because of the phase transition behavior of these polymers, and therefore these formulations are easy to pour or load. Results are presented for separation of double‐stranded DNA fragments (< 2000 bp) in capillary, tube, and slab electrophoresis. Preliminary results are also presented for separation of single‐stranded sequencing fragments by capillary electrophoresis. In the tube format, good resolution was obtained for ΦX174/HaeIII fragments. In the slab format, excellent resolution of double‐stranded DNA was obtained, including simulaneous separation of a 10 bp ladder up to 150 base pairs. In the capillary format, pBR322/MspI fragments were completely resolved, and single‐base resolution of sequencing fragments was obtained up to 150 bases. We believer that temperature‐sensitive polymers represent a new and promising class of electrophoretic media.
Comment on mobility‐shift computations featuring cage effects
Authors: Cann John R. 
Published: May 2005

AbstractTheoretical mobility‐shift patterns are computed by solution of conservation equations for electrophoresis coupled with chemical reaction. The chemical reaction term is often formulated in terms of dissociation of the protein‐DNA complex in a gel cage. This formulation assumes that once the dissociated protein escapes the cage, it goes down a sink and is totally lost. This implies that the concentration of the escaped protein is too low to affect sign ficantly the rates of protein‐DNA association along its migration pathway.
Three novel mutations of the NF1 gene detected by temperature gradient gel electrophoresis of exons 5 and 8
Authors: Horn Denise , Robinson Peter N. , Böddrich Annett , Buske Annegret , Tinschert Sigrid , Nürnberg Peter 
Published: May 2005

AbstractWe screened a total of 100 unrelated patients with neurofibromatosis type 1 (NF1) for mutations in exons 5 and 8 of the NF1 gene using temperature gradient gel electrophoresis (TGGE). Careful interpretation of exon 5 TGGE patterns was necessary due to interference by an exonic polymorphism. Three novel mutations were identified: a stop mutation in exon 5 (Q239X) caused by a C→T transition at cDNA nucleotide position 715, a transition at the invariant G of the splice accceptor site in intron 4c (G655‐1A), and a transversion at the invariant G of the splice donor site in intron 8 (G1185+1T). Analysis of mRNA revealed the predicted abnormal splice products. While skipping of exon 5 causes a shift in the reading frame with a premature stop codon downstream in the middle of exon 6, skipping of exon 8 leads to an in‐frame deletion with the predicted protein product being shortened by 41 amino acids.
Separation mechanism of pullulan solution‐filled capillary electrophoresis of sodium dodecyl sulfateproteins
Authors: Nakatani Manabu , Shibukawa Akimasa , Nakagawa Terumichi 
Published: May 2005

AbstractThe separation mechanism of capillary electrophoresis of sodium dodecyl sulfate (SDS)‐proteins using pullulan with a molecular mass range of 50 000–100 000 as a separation matrix was investigated. The pullulan solution was filled into fused‐silica capillaries whose inner walls were coated with linear polyacrylamide through chemically stable Si‐C linkages. Baseline separations of SDS proteins were achieved at concentrations ranging from 3–10% w/v of pullulan. The entanglement threshold of pullulan solution was found to be around 0.5% w/v, indiacating migration of SDS‐proteins through an entangled pullulan network. Ferguson plots exhibited a linear relationship between log mobility and pullulan concentration. Linear relationships were also obtained for double logarithmic plots of the electrophoretic mobility and protein molecular mass. These results show that the separation is based on mass discrimination in accordance with the Ogston theory.