bluuebunny/crossref_metadata_2025 huggingface dataset:
Transverse pore gradient gel electrophoresis, using the PhastSystem
Authors: Buzás Zsuzsanna , Wheeler David L. , Garner Mark M. , Tietz Dietmar , Chrambach Andreas
Published: May 2005

The application of pore gradient gels prefabricated for the PhastSystem (Pharmacia) to transverse pore gradient gel electrophoresis is demonstrated. It has the twofold advantage of (i) horizontal positioning, avoiding gel stretching during the preparation of these gels and resulting pore size irreproducibility experienced with vertically applied pore gradient gels, which necessitate an orthogonal transfer of spacers, and (ii) miniaturized gel dimensions, which allow a small sample load and a short duration of electrophoresis and staining.
The determination of cocaine and related substances by micellar electrokinetic capillary chromatography
Authors: Trenerry V. Craige , Robertson James , Wells Robert J.
Published: May 2005

A quantitative method for the determination of cocaine and related substances by micellar electrokinetic capillary chromatography (MECC) is described. Quantitative results obtained for both monitor materials and a number of actual drug seizures by this new capillary electrophoretic method are comparable to a gas chromatography (GC) run in parallel, both in the values and coefficient of variation achieved. The advantages of this new method include minimal use of solvents, the ability to readily automate the procedure and the ability to quantitate illicit heroin seizures under the same conditions with the exception of detector wavelength alteration. The method has proved rugged and reliable for both heroin and cocaine in a number of inter‐laboratory proficiency studies.
Immobilized pH gradients: New pK values of acrylamido buffers in poly(N‐acryloylaminoethoxyethanol) matrices
Authors: Bossi Alessandra , Righetti Pier Giorgio , Chiari Marcella
Published: May 2005

A novel matrix consisting of N‐acryloylaminoethoxyethanol, a hydrophilic monomer extremely resistant to hydrolysis, was recently reported by Chiari et al. (Electrophoresis, 1994, 15, 177–186). When using it as a matrix for grafting immobilized pH gradients for isoelectric focusing, a shift in protein spot position was noticed. This was attributed to a shift in pK values of the Immobiline buffers when changing from the standard poly(acrylamide) to a poly(N‐acryloylaminoethoxyethanol) matrix. A series of 1 pH unit gradients was constructed, where a single buffering Immobiline was used and titrated with a counterion having a pK removed by at least 3 pH units from the nearest extreme of the generated pH interval. It was noted that all compounds became weaker acids and bases, with a ΔpK ranging from −0.02 to −0.06 for the acids (pK 3.6, 4.4, and 4.6) and a ΔpK ranging from −0.12 tp −0.20 for the bases. The new pK values for the seven commercially available buffers are thus pK 3.6 ‐> pK 3.58, pK 4.4 ‐> pK 4.36, pK 4.51 ‐> 4.45, pK 6.21 ‐> 6.09, pK 7.06 ‐> 6.94, pK 8.50 ‐> pK 8.37, and pK 9.59 ‐> pK 9.39. These values refer to 10°C in the gel phase, the first value in poly(acrylamide) and the second in poly(N‐acryloylaminoethoxyethanol).
Quantitative studies of different injection systems in capillary electrophoresis
Authors: Ermakov Sergey V. , Zhukov Michael Yu , Capelli Laura , Righetti Pier Giorgio
Published: May 2005

For a rigorous assessment of the precise amount of sample loaded, for quantitation purposes, different sample injection systems were evaluated with two commercially available units, Waters Quanta 4000 and Beckman P/ACE 2100. In the first system, sample introduction by hydrostatic means (i.e., placing the sample vial at some height, usually 10.1 cm, above the other capillary end) was evaluated. It was found that in this system there is a constant positive bias, i.e. the amount of sample loaded lies on a curve parallel and above the theoretically predicted loading curve. However, the excess of mass loaded was constant along the injection times explored (covering from 5 to 35 s) and, for a 75 μm capillary, was found to be of the order of +6 nL (above the expected injected value). Thus it is easy to correct for this sample bias. In the electrokinetic mode, a very good correlation between expected and predicted sample loads was obtained for both units. In the pressure system (by positive pressure from a nitrogen tank, Beckman unit) a substantial discrepancy was found between experimental and predicted values (13.5% overload). Since the manufacturer claims a constant pressure of 0.5 psi, i.e. 3447.5 Pa, it would appear that the injection pressure is higher than the given value. Other causes for variation in sample load (e.g., as caused by diffusion of sample in the capillary just prior to injection, by the sudden insertion of the capillary tip into the sample vial, etc., lumped together in a general term as “extraneous injection”) have been evaluated and found to contribute to an additional sample volume injection of the order of 1–2 nL, i.e., quite negligible. In conclusion, it is felt that both the hydrostatic and electrokinetic injection modes are highly reliable. The positive pressure mode can fail due to leakage of O‐rings, valve malfunctioning and to the typical problems of mechanical systems. Thus, the latter injection system should be checked frequently for potential mechanical failures.
Separation of lipophilic compounds by micellar electrokinetic chromatography with organic modifiers
Authors: Otsuka Koji , Higashimori Mitsuo , Koike Ryo , Karuhaka Kaoru , Okada Yukihiro , Terabe Shigeru
Published: May 2005

Separation of lipophilic compounds such as polyaromatic hydrocarbons (PAH) by micellar electrokinetic chromatography (MEKC) with organic modifiers was investigated. Dimethyl sulfoxide (DMSO) and acetone were used as organic modifiers, and sodium dodecyl sulfate (SDS) as a surfactant or a micelle forming reagent. By using 25 mM SDS (pH 7.0), containing 50% v/v DMSO, 8 PAHs were separated. Similarly, with 25 mM SDS, containing 30% acetone, 13 PAHs were successfully separated. For the calculation of thermodynamic quantities, critical micelle concentrations of SDS in buffers containing DMSO or acetone were measured.
Spatial and temporal depletion of ions from noncrosslinked denaturing polyacrylamide in capillary electrophoresis
Authors: Figeys Daniel , Renborg Annika , Dovichi Norman J.
Published: May 2005

Electrical conductivity across a polyacrylamide‐filled capillary decreases during the separation of DNA sequencing fragments. This conductivity decrease is localized to the first few centimeters at the injection (negative) end of the capillary; no conductivity change is noted at the detection (positive) end of the capillary. The zone of decreased conductivity extends further into the capillary as the separation proceeds. The zone is most important for freshly prepared capillaries; capillaries used nine days after polymerization generate an insignificant current drop. The data are consistent with ionic depletion due to differences in transport numbers between the separation medium and the buffer reservoirs.
Hybridization of the variable number of tandem repeat core oligonucleotide GNNGTGGG to 350 cosmids from chromosome 7
Authors: Melmer Georg
Published: May 2005

An efficient method using short oligonucleotides is described for the isolation of minisatellite markers of the type variable number of tandem repeats (VNTR). It is used to screen 350 cosmids from chromosome 7 for the presence of such potentially polymorphic DNA segments. From the number of hybridization signals to chromosome 7 cosmids, I estimate the human genome to have at least about 15000 VNTR loci containing minisatellite core sequences, which is well above previous estimates.
Capillary zone electrophoresis of DNA fragments in a novel polymer network: Poly(N‐acryloylaminoethoxyethanol)
Authors: Chiari Marcella , Nesi Marina , Righetti Pier Giorgio
Published: May 2005

Migration of DNA fragments in the 51 to 21 226 bp size range has been investigated by capillary zone electrophoresis in entangled solutions of linear poly(N‐acryloylaminoethoxyethanol) from 8 to 16%. Plots of log mobility vs. log size (in base pairs, bp) clearly evidence three different migration regimes: according to Ogston (i.e. as spherical globules) up to 200 bp, reptation without stretching up to 3–4000 bp and reptation with partial stretching for larger fragments. Guidelines on the % polymer to be used for optimum resolution can be obtained from plots of peak spacing (in seconds per base pair) vs. % of polymer in solution and from standard plots of peak resolution vs. % polymer. An optimum linear poly(N‐acryloylaminoethoxyethanol) concentration, allowing for good resolution of most fragments, from small to large, is found in the range 10% to 12% polymer (as opposed to conventional polyacrylamide, where a sharp maximum is obtained at 6% polymer). It is hypothesized that in situ polymerization allows for formation of a large distribution of polymer sizes, thus facilitating simultaneous separation of short and long DNA fragments based on the principle that shorter polyacrylamide chains will sieve shorter DNA fragments and vice versa. Due to the fact that the novel monomer is 500 times more resistant to hydrolysis and more hydrophilic than acrylamide, its polymeric solutions allow repeated sample injections (> 35) with minimal absolute standard deviations (of the order of ± 3%). No carryover nor sample precipitation at the injection port is experienced with the new matrix composed of poly(N‐acryloylaminoethoxyethanol).
Purification of human recombinant superoxide dismutase by isoelectric focusing in a multicompartment electrolyzer with zwitterionic membranes
Authors: Wenisch Elisabeth , Vorauer Karola , Jungbauer Alois , Katinger Hermann , Righetti Pier Giorgio
Published: May 2005

Human recombinant superoxide dismutase (SOD), purified to homogeneity, is resolved by both conventional isoelectric focusing and immobilized pH gradients into three bands, with isoelectric points (p/s) in the pH range 4.8 to 5.1, the pI 4.80 form representing the minor component. Due to the fact that this enzyme is expressed in E. coli, N‐terminal acetylation or glycosylation should be ruled out. When purified by small‐scale preparative isoelectric focusing in immobilized pH gradient gels, it was found that, upon subsequent analysis, the pI 5.07 form would band in the same position, but the intermediate pI 4.92 band would split into the upper (pI 5.07) and the lower (pI 4.80) species, in nearly the same amounts, whereas the lowest pI component would always generate both the intermediate and upper forms. Enzymatic essays pointed out that these three isoforms had nearly the same specific activity, slightly higher than that of the starting material. Metal analysis indicated that all three forms contained the same metal/protein ratio, approaching the value Cu2Zn2‐SOD, as reported in the literature. Circular dichroism spectra of the pI 4.80 and 5.07 forms showed the same profile in the 190–240 nm range, but marked differences in the 250–350 nm region. Treatment with EDTA produces 1–2 additional, slightly higher pI isoforms, whereas treatment with KCN generates a number of higher pI components, reaching pI values as high as pH 7, with nearly complete disappearance of the three major SOD isoforms. It is concluded that these three isoforms could represent interconvertible species, the highest pI component representing the most stable conformer.
Sample preparation of human tumors prior to two‐dimensional electrophoresis of proteins
Authors: Franzén Bo , Hirano Takashi , Okuzawa Ken , Uryu Kazuto , Alaiya Ayodele A. , Linder Stig , Auer Gert
Published: May 2005

Sample preparation procedures are critical for obtaining high quality two‐dimensional electrophoresis (2‐DE) separations from clinical tumor materials. In this communication, we describe procedures which we have found satisfactory for preparing various tumor samples for 2‐DE. By these procedures, tumor cells are enriched from serum proteins and contaminating stromal cells. Tumors can be prepared with good reproducibility to obtain high resolution using these techniques.
High resolution one‐dimensional electrophoretic separation and partial characterisation of human head hair proteins
Authors: Khawar Sitwat Lubna , Watson Kenneth , Jones Graham L.
Published: May 2005

A reproducible, rapid procedure for the extraction, labelling and separation of human hair proteins by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) has been developed. Hair proteins were extracted in 8 M urea, containing 0.2 M mercaptoethanol, followed by sonication. Extracts were neutralised with Tris and incubated with either labelled (14C) or unlabelled iodoacetic acid to S‐carboxymethylate cysteine groups. Proteins were separated on 12.5% SDS‐polyacrylamide gels and gels stained with Coomassie Brilliant Blue and/or silver nitrate to reveal major protein bands. Gels were then treated with a fluorographic agent, dried and autoradiographed to reveal major sites of S‐carboxymethylation. A given gel was scanned by laser densitometry after Coomassie and/or silver stain to quantitate the protein content of each major protein zone. An autoradiogram of the same gel was scanned to estimate the cysteine content of each major zone. In this way it was possible to partially characterise rapidly and reproducibly many different protein zones in different individual samples on one gel at the same time. By calculating the ratio of autoradiograph absorbance to Coomassie Blue absorbance, protein zones could be assigned to four different categories, viz: very high cysteine (VHC) proteins, high cysteine (HC) proteins, low cysteine (LC) proteins and very low cysteine (VLC) proteins. The method described is reproducible, rapid and inexpensive enough to be suitable for mass screening. Overall the results were more informative than previously reported one‐dimensional separations and indeed this technique may well be more suited to forensic and/or population investigations than the much more laborious and time‐consuming two‐dimensional techniques.
Characterizing allelic proteins for genome mapping in maize
Authors: Touzet Pascal , Morin Catherine , Damerval Catherine , Guilloux Martine Le , Zivy Michel , De Vienne Dominique
Published: May 2005

Mapping cDNA probes in order to construct genetic linkage maps is becoming a widespred strategy for genome analysis and gene isolation, particularly in cultivated plant species. Nevertheless, almost all cDNAs reveal two or more unlinked loci, making it difficult to identifiy the gene(s) actually expressed. In a highly polymorphic species, such as maize, two‐dimnsional polyacrylamide gel electrophoresis (2‐D PAGE) of proteins may circumvent this limitation. With the analysis of various segregating populations, we previously showed that the apparent position shifts of proteins on the 2‐D gels are monogenic and codominant. In this paper we compared allelic proteins on the basis of their high performance liquid chromatography (HPLC) profile and partial amino acid sequences. In a sample of 20 position shifts, the allelic proteins appeared to be similar in all but one case, strongly suggesting that polymorphism of structural genes is involved. Thus 2‐D PAGE could prove to be a useful tool for genome mapping: when a cDNA probe detects several loci, a position shift of the encoded protein will allow the identification of the gene translated in the organ considered.
Polymorphic human specific Alu insertions as markers for human identification
Authors: Novick Gabriel E. , Novick Corina C. , Yunis Juan , Yunis Emilio , Martinez Kianfa , Duncan George G. , Troup Gary M. , Deininger Prescott L. , Stoneking Mark , Batzer Mark A. , Herrera Rene J.
Published: May 2005

Alu sequences represent the largest family of short interspersed repetitive elements (SINEs) in humans with 500 000 copies per genome. Recently, one Alu subfamily was found to be human specific (HS). We originally described the use of polymorphic HS Alu insertions as a tool in population studies and recently as tools in DNA fingerprinting and forensic analysis. In this report, we will use this simple polymerase chain reaction (PCR) base technique for the detection of HS Alu insertion polymorphisms. We will test the resolving power of this DNA profiling approach in both population genetics and paternity assessment. At the population level, we will describe the genotypic distribution of five polymorphic Alu insertions among 3 populations from the American continent, one of African origin, the other two Amerindians. Insight into their relationships will be provided. At the family level, we will examine one European American family of seven individuals and the same pedigree will also be characterized by way of the two systems currently and widely used to ascertain paternity: PCR‐sequence specific oligonucleotide probe hybridization (PCR‐SSO) and PCR‐restriction fragment length polymorphism (PCR‐RFLP) of human leucocyte antigen (HLA) class II molecules, and a standard RFLP protocol used in forensic casework and paternity studies. The importance and strengths of the method as well as its perspectives for future use in filiation studies will be evaluated.
Genotyping of human deoxyribonuclease I polymorphism by the polymerase chain reaction
Authors: Yasuda Toshihiro , Nadano Daita , Tenjo Etsuko , Takeshita Haruo , Sawazaki Kazumi , Nakanaga Masao , Kishi Koichiro
Published: May 2005

We recently completely elucidated the molecular basis of genetic polymorphism in human deoxyribonuclease I and found it to be controlled by four codominant alleles, DNASE1*1, *2, *3 and *4. In this paper we describe a novel DNase I‐genotyping system that could be used directly on DNA samples using the polymerase chain reaction (PCR) based on the three nucleotide substitutions underlying the protein polymorphism. The system consists of three independent reactions. Since the substitutions neither suppress nor create any known enzyme recognition site in the DNase I gene, two separate mismatched PCR followed by XhoI digestion methods were introduced to discriminate between the DNASE1*1 (or *3) and the DNASE1*2 (or *4) alleles, and to detect the DNASE1*4 allele. An amplification refractory mutation system was employed to detect DNASE1*3. A 100% correlation was found between the results of this genotyping method and those obtained by phenotyping using conventional isoelectric focusing. The high sensitivity and specificity of this genotyping method allows us to survey DNase I‐polymorphism in small DNA samples.
Lung tumor cells: A multivariate approach to cell classification using two‐dimensional protein pattern
Authors: Schmid Hans‐Rudolf , Schmitter Doris , Blum Philippe , Miller Mark , Vonderschmitt Dieter
Published: May 2005

High resolution two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) is a powerful research tool for the analytical separation of cellular proteins. The qualitative and quantitative pattern of polypeptides synthesized by a cell represents its phenotype and thus defines characteristics such as the morphology and the biological behavior of the cell. By analyzing and comparing the protein patterns of different cells it is possible to recognize the cell type and also to identify the most typical features of these cells. In applied pathology it is often difficult to identify the tissue of origin and the stage or grade of a neoplasia by cellular morphology analyzed by classical or immunostaining procedures. The protein pattern itself is the most characteristic feature of a cell and should therefore contribute to the identification of the cell type. For this reason we separated protein fractions originating from different lung tumor cell lines using 2‐D PAGE and we compared the resulting patterns on a multivariate statistical level using correspondence analysis (CA) and ascendant hierarchical clustering (AHC). The results indicate that (i) protein patterns are highly typical for cells and that (ii) the comparison of the protein patterns of a set of interesting cell types allows the identification of potentially new marker proteins. 2‐D PAGE is thus a unique and powerful tool for molecular cytology or histopathology, unveiling the protein expression level of tissues or cells.
A spot cloning method for restriction landmark genomic scanning
Authors: Ohsumi Tomoya , Okazaki Yasushi , Hirotsune Shinji , Shibata Hideo , Muramatsu Masami , Suzuki Harukazu , Taga Chiharu , Watanabe Sachihiko , Hayashizaki Yoshihide
Published: May 2005

We introduce two new methods for target cloning of DNA fragments corresponding to spots on the two‐dimensional profile of restriction landmark genomic scanning (RLGS). One is a restriction trapper‐based method and the other is a polymerase chain reaction (PCR) mediated method. Both are designed to select the target DNA fragments from a large amount of unlabeled background DNA fragments in the RLGS gel which produce background clones. The restriction trapper method is simple, with a cloning efficiency that is not biased by the length of the target DNA nor by its GC content. On the other hand, the PCR‐mediated method is efficient for cloning DNA fragments from a small amount of starting materials. These methods provide us with powerful tools for isolating DNA clones identified by the RLGS system as interesting spots. This paper reports the precise protocols of these methods and discusses their application and usefulness.
The use of restriction landmark genomic scanning to scan the mouse genome for endogenous loci with imprinted patterns of methylation
Authors: Shibata Hideo , Yoshino Kiyoshi , Muramatsu Masami , Plass Christoph , Chapman Verne M. , Hayashizaki Yoshihide
Published: May 2005

Restriction landmark genomic scanning (RLGS) has been used to screen endogenous loci for imprinted patterns of methylation. The screening method is based upon the identification of genetic variation in RLGS profiles between different strains and determining whether specific variant landmarks are transmitted equally to the progeny of reciprocal F1 matings. The RLGS profiles of C57BL/6 (B6) and DBA/2 (D2) and their reciprocal hybrids were produced with two enzyme combinations that used NotI as the landmark enzyme and two combinations that used BssHII. An estimated 13% of the spots are either B5‐ or D2‐specific in these tests, giving a total of nearly 1000 variant loci that were examined for imprinted methylation. Three candidate loci for imprinted regulation were identified in these analyses. We also used crosses of more genetically diverse parents to increase the number of variant loci screened. Interspecific crosses of B6 with the M. musculus strain PWK and intrasubspecific crosses between B6 and the M. molossinus strain MSM expanded the levels of variation between the parental strains in the cross to an estimated 31% and 26%, respectively. The RLGS patterns for one NotI combination and one BssHII profile were examined for each of these crosses, giving approximately 2000 additional loci that were screened for imprinted patterns of methylation. Eight loci with imprinted patterns of transmission were observed out of 3040 loci tested. The chromosomal locations for the three B6 and D2 specific loci, Irlgs 1–3, were identified using BXD recombinant inbred strain analysis. Irlgs 1 and 3 are B6‐ and D2‐specific loci that had the same strain distribution pattern which mapped to the central region of chromosome 9. Irlgs 2 (U2afbp‐rs) was mapped to the proximal region of chromosome 11, which was reported as an imprinted region identified with uniparental disomy mice. An imprinted gene, U2 auxiliary factor binding protein‐related sequence (U2afbp‐rs), was identified for Irlgs2 locus, which encoded 51 kDa protein that had significant homology to the human U2af 35 kDa small subunit.
Enhancing protein solubilization with nondetergent sulfobetaines
Authors: Vuillard Laurent , Marret Nathalie , Rabilloud Thierry
Published: May 2005

A significant increase in the yield of protein extraction by the addition of sulfobetaine‐type mild solubilization agents is shown for microsomal membrane proteins and proteins from lyophilized platelets, with a maximum increase in yield of up to 100%. These agents are also applied to native isoelectric focusing in immobilized pH gradients. The absence of significant denaturation induced by these solubilizers is shown for creatine kinase.
A highly discriminating octoplex short tandem repeat polymerase chain reaction system suitable for human individual identification
Authors: Oldroyd Nicola J. , Urquhart Andrew J. , Kimpton Colin P. , Millican Emma S. , Watson Stephanie K. , Downes Teresa , Gill Peter D.
Published: May 2005

Through the use of fluorescence‐based polymerase chain reaction systems, a highly discriminating multiplex with the potential for individual identification has been developed. The use of multiple dye technology enabling loci with overlapping size ranges to be co‐amplified has enabled us to successfully amplify seven tetranucleotide short tandem repeat loci within a single reaction in a discriminating power in the region of 1 × 109. Three out of the seven loci employed exhibit alleles differing in size by only 2 bp as opposed to the conventional 4 bp, which results in such loci being more powerful in terms of distinguishing between samples, particularly when co‐amplified in this manner. The size ranges of the loci contained within the system are such that windows still exist for the inclusion of additional loci at a later stage, which could increase the discriminating power of the system still further. In addition, further weight and utility is lent to the system through the incorporation of a simple and reliable sex test involving the amplification of a segment of the X–Y homologous gene Amelogenin.
Analysis of underivatized amino acids by capillary electrophoresis using constant potential amperometric detection
Authors: Guo Yong , Colón Luis A. , Dadoo Rajeev , Zare Richard N.
Published: May 2005

A mixture of native (underivatized) amino acids is separated by capillary electrophoresis under alkaline conditions (pH ≈︁ 12) and amperometrically detected with a copper‐disk microelectrode. A simple design facilitates capillary‐electrode alignment without the need for micropositioning equipment. The limits of detection for the amino acids are in the low μm concentration range, and the signal response is linear over 2–3 orders of magnitude. This procedure is applied to analyze the amino acid hydrolysis products from cytochrome c.
Sensitivity enhancement and second‐dimensional information from solid phase extraction‐capillary electrophoresis of entire high‐performance liquid chromatography fractions
Authors: Strausbauch Michael A. , Madden Benjamin J. , Wettstein Peter J. , Landers James P.
Published: May 2005

A novel separation technique is demonstrated for peptide analysis of entire 100 μL high‐performance liquid chromatography (HPLC) fractions in a single, low pH, capillary zone electrophoresis (CZE) separation. The method employs a hybrid capillary consisting of a removable inlet section containing a reverse‐phase packing and a standard CZE bare‐fused silica separation capillary. The packed tip allows sample to be concentrated at flow rates greater than 20 μL/min and released in a controlled manner for CZE separation. Separations are comparable to standard CZE with minor modifications in selectivity. Using a mixture of model peptides, the hybrid capillary is shown to effect enhancement in sensitivity of ≥ 100‐fold. Five fractions from the HPLC separation of peptides obtained from the digestion of bovine serum albumin (BSA) were analyzed by the solid phase extraction‐capillary electrophoresis (SPE‐CE) method which was shown to provide rapid second‐dimensional information. Practical concentration limits of detection for peptides are calculated to be 1‐‐10 ng/mL with this technology.
Extended theory of the electrophoretic mobility‐shift analysis of nonspecific protein‐DNA complexes, featuring cooperativity
Authors: Coombs Robert O. , Cann John R.
Published: May 2005

The simulated electrophoretic mobility‐shift behavior of a model system, in which the nonspecific binding of a protein to a DNA fragment is cooperative, was compared with the experimental behavior of the DNA: histone‐like bacterial protein (HU) system. It was concluded that the binding of HU to an 88 bp DNA fragment is, at least, not highly cooperative. The theory of mobility‐shift analysis was extended even further to encompass high affinity sequence‐specific binding of protein to a DNA fragment followed by weak nonspecific binding, the latter governed by conditional probabilities. In addition to featuring a ladder of incremental protein‐DNA complexes, the computed mobility‐shift patterns placed emphasis upon stabilization of weak, nonspecific complexes in gel cages.
Variation of the pH of the background electrolyte due to electrode reactions in capillary electrophoresis: Theoretical approach and in situ measurement
Authors: Corstjens Hugo , Billiet Hugo A. H. , Frank Johannes , Luyben Karel C. A. M.
Published: May 2005

Electrode reactions during the electrophoretic process may change the pH of the buffer and subsequently the migration behavior of solutes with resultant loss of reproducibility. A theoretical treatment of pH variations due to electrolytic processes is presented. The choice of buffer appears to have a dramatic influence on the pH variations observed, even if substantial buffer action is expected at the pH chosen. The experimental evaluation of the separation of 4‐hydroxy‐3‐methoxycinnamic acid and 3‐hydroxybenzoic acid reveals that the quality of the separation decreases continuously from a baseline separation observed in the first experiment to a comigration of the two solutes (resolution = 0) in the ninth experiment. A pH decrease of about 0.05 pH units accounts for the observed changes in mobility.

A novel in situ pH measurement approach is presented, in which the mobility, peak area, and peak height of an indicator dye are related to the pH in the capillary. This enables the identification and quantification of pH variations during electrophoretic runs: the pH decreases at the anodic side already after the first experiment and pH variations as small as 0.02 pH units can be measured. The variations in peak height appear to be less suited. The calculated pH variations are in close agreement with the ones obtained experimentally.
Toxicant‐induced alterations in two‐dimensional electrophoretic patterns of hepatic and renal stress proteins
Authors: Witzmann Frank A. , Fultz Carla D. , Lipscomb John C.
Published: May 2005

Recent studies in this laboratory and by others suggest that two‐dimensional polyacrylamide gel electrophoresis of proteins (2‐DE) possesses significant utility in the detection of chemical toxicity and in providing information regarding toxic mechanism. After having identified a set of specific heat‐shock and glucose‐regulated proteins whose expression in rodent liver and kidney is highly conserved and constitutive, we compared the effect of in vivo exposure to perfluoro‐n‐octanoic acid and perfluoro‐n‐decanoic acid on their expression. The following stress proteins were identified, their x, y coordinate positions mapped, and abundance statistically analyzed and compared: hsp32, hsp60, hsc70, hsp70, hsp90, grp75, grp94, protein disulfide isomerase (PDI), and ER60. We report here that the stress response to perfluorocarboxylic acids is tissue‐, toxicant‐, and stress protein class‐specific and dose‐related. Furthermore, because nearly all of the proteins studied were constitutively expressed at detectable levels in both liver and kidney, the 2‐DE stress protein pattern may be suitable to future toxicologic screening applications.
Quantitative analysis of hyaluronan in human synovial fluid using capillary electrophoresis
Authors: Grimshaw James , Trocha‐Grimshaw Jadwiga , Fisher William , Rice Aislinn , Smith Susan , Spedding Peter , Duffy John , Mollan Raymond
Published: May 2005

The glycosaminoglycan, hyaluronan, can be detected in human synovial fluid by capillary electrophoresis (CE). Variations in peak shape make this technique unsuitable for quantitative analysis of hyaluronan in raw synovial fluid. Quantitative analysis was achieved by hydrolysis of the polymeric hyaluronan to the tetrasaccharide by the action of testicular hyaluronidase and separation of the product using CE. A UV detector operating at 200 nm was used. The X‐ray contrast material, omnipaque, a propriety aqueous solution of iohexol was used as internal standard. A second peak in the electropherogram of synovial fluid was quantified. The variation in concentrations of these two components correlate with the arthritic disease state of a joint.
High‐speed separation of subnanomolar concentrations of noradrenaline and dopamine using capillary zone electrophoresis with laser‐induced fluorescence detection
Authors: Bert Lionel , Robert Frédéric , Denoroy Luc , Renaud Bernard
Published: May 2005

Capillary zone electrophoresis with laser‐induced fluorescence detection has been shown to give rapid separations with high resolution and sensitivity. These advantages are documented for catecholamines analysis in the present work, which shows that separation of subnanomolar concentrations of dopamine and noradrenaline (detection limits of 8.6 × 10−11 M, corresponding to a detected amount of 143 zeptomoles of each catecholamine) can be performed in 82 s with an efficiency of several million theoretical plates.
Novel acrylamido monomers with higher hydrophilicity and improved hydrolytic stability: III. DNA separations by capillary electrophoresis in poly(N‐acryloylaminopropanol)
Authors: Gelfi Cecilia , Simò‐Alfonso Ernesto , Sebastiano Roberto , Citterio Attilio , Righetti Pier Giorgio
Published: May 2005

Separation of DNA fragments in a novel polymer network, consisting of N‐acryloylaminopropanol (AAP) is reported. The performance of this novel monomer, as a sieving liquid polymer in capillary zone electrophoresis, was evaluated. In 50 μm ID capillaries, an 8% solution of poly(AAP) can afford apex‐resolution of the 123/124 bp adjacent pair of DNA fragments in marker V, typically unresolved in any poly(acrylamide) formulation. It is proposed that the distal –OH group in the AAP molecule can form transient H‐bonds with the DNA double helix. Molecular modeling shows a meandering structure for poly(AAP), lacing the walls of half a cylinder, with kinks protruding at regular intervals, potentially able to fit into the major groove of DNA. Contrary to previously held beliefs, there seems to be a minimum length of the polymer for proper sieving of DNAs. For poly(acrylamides), a weight average molecular mass Mw 30 000 polymer offers no resolution, whereas a polymer of 250 000 to 400 000 Da exhibits optimum resolving power. For poly(AAP), the optimal length is in excess of 450 000 Da in Mw. Thus, it is shown that both the chemical composition of the monomer and the length of the polymer play a subtle, cooperative role in DNA separation.
The relative contributions of dispersion and diffusion to band spreading (resolution) in gel electrophoresis
Authors: Yarmola Elena , Sokoloff Helen , Chrambach Andreas
Published: May 2005

DNA of approximately 2 kbp in length was previously found not to diffuse significantly in 1–1.5% agarose gels in the absence of an electric field, but to disperse during electrophoresis (Yarmola, E., Chrambach, A., Electrophoresis 1995, 16, 345–349). Accordingly, a process distinct from diffusion, and responsible for band spreading with migration time in gel electrophoresis, was defined as dispersion. Correspondingly, the diffusion coefficient, D(diff), was distinguished from a dispersion coefficient, D(disp). For DNA of approximately 1, 2 and 3 kbp, D(diff) and D(disp) were measured in agarose gel electrophoresis (1.0% SeaKem GTG). In that order of DNA length, D(disp) / D(diff) was found to increase from 5 to 15 to 45, showing that with increasing DNA length, time‐dependent band spreading, and thus resolution in gel electrophoresis, is governed predominantly by dispersion, not diffusion. It is assumed that the essential part of electrophoretic dispersion is due to entanglement of the DNA molecule in the gel. Indirect evidence for such an entanglement derives from the observation of peak asymmetry and its interpretation by the Giddings‐Weiss model.
Rapid loading of large sample volumes, analyte cleanup, and modified moving boundary transient isotachophoresis conditions for membrane preconcentration‐capillary electrophoresis in small diameter capillaries
Authors: Tomlinson Andy J. , Benson Linda M. , Jameson Stephen , Naylor Stephen
Published: May 2005

Using a removable membrane preconcentration (mPC) cartridge, large sample volumes can be loaded prior to final assembly of the mPC capillary electrophoresis (CE) capillary. For narrow‐bore (⩽ 25 μm ID) uncoated mPC‐CE capillaries, applied to peptide analysis, efficient moving boundary transient isotachphoresis (tITP) conditions at the onset of electrophoresis are described. The enhancement of mPC‐CE‐mass spectrometry (MS) technology afforded by rapid sample loading and modified moving boundary tITP conditions are demonstrated by analysis of major histocompatibility complex (MHC) class I peptides that were derived from a Kb precipitation of mouse EL‐4 cells. Furthermore, we demonstrate the structural characterization of these immunologically significant molecules by mPC‐CE‐tandem mass spectrometry (mPC‐CE‐MS/MS).
A kinetic study of the methanolysis of the sulfonylureas bensulfuron methyl and sulfometuron methyl using capillary electrophoresis
Authors: Matchett William H. , Winnik Witold , Betowski Leon O. , Brumley William C.
Published: May 2005

The instability of sulfonylureas in solution in methanol has led us to a kinetic study of methanolysis of two sulfonylureas using capillary electrophoresis. In a preliminary experiment, solutions of the seven compounds, bensulfuron methyl, sulfometuron methyl, nicosulfuron, chlorimuron ethyl, thifensulfuron methyl, metsulfuron methyl, and chlorsulfuron were prepared in methanol and in acetonitrile. After six weeks at room temperature the compounds dissolved in acetonitrile were quite stable as shown by electropherograms obtained with free zone capillary electrophoresis (CE). All seven of the compounds dissolved in methanol had undergone extensive degradation and in some cases the sulfonylurea could not be detected. Bensulfuron methyl and sulfometuron methyl were selected for further detailed study. Solutions of these two compounds in methanol were heated at 5, 40, 50, 57, and 65°C. No decomposition of either compound was observed in similar solutions of acetonitrile incubated at 57°C. The rates of decomposition in methanolic solutions were estimated from the decrease in instrumental response representing the peaks of the two sulfonylureas. The methanolysis of both compounds proceeded with pseudo first‐order kinetics as evidenced by the fact that semilogarithmic plots of the concentration of the substrate as a function of time were linear (correlation coefficients of 0.99). Rate constants and half‐lives for the reactions at 40, 50, 57, and 65°C were determined. These varied by factors of 18 to 22 depending upon the reaction temperature. The energies of activation for these reactions were estimated from the appropriate Arrhenius plots and found to be 26 kcal per mole for bensulfuron methyl and 24.5 kcal per mole for sulfometuron methyl. The kinetics of appearance of the products of methanolysis were directly related to and accurately predicted by the pseudo first‐order rate constants observed for disappearance of the sulfonylureas. Kinetic study of the reaction mixture by negative and positive‐ion mass spectrometry indicated that the products of methanolysis of sulfometuron methyl were 2‐carboxymethyl (N‐carboxymethyl)benzsulfonamide and 2‐amino‐4,6‐dimethyl pyrimidine and suggested a general mechanism for methanolysis of each of the seven compounds studied.
Prefractionation of protein samples prior to two‐dimensional electrophoresis
Authors: Corthals Garry L. , Molloy Mark P. , Herbert Ben R. , Williams Keith L. , Gooley Andrew A.
Published: May 2005

Thousands of proteins may be visualised on a two‐dimensional (2‐D) gel, but only hundreds are present at levels sufficient for chemical analysis. Therefore, prefractionation of protein samples prior to 2‐D polyacrylamide gel electrophoresis (PAGE) will be important for the investigation of proteins that are present at sub‐picogram levels in physiological samples. We describe an approach to prefractionate protein samples prior to 2‐D PAGE using the Gradiflow, which is a new (preparative) electrokinetic membrane apparatus designed to fractionate proteins in a number of different ways. We have fractionated human serum under nonreducing conditions using the ‘reflux’ mode, in which proteins are fractionated according to their relative mobility under controlled electrophoretic conditions, where the current is periodically reversed. We describe how fractionation occurs and present examples of enrichment of specific proteins.
Probing protein function using a combination of gene knockout and proteome analysis by mass spectrometry
Authors: Dainese Paola , Staudenmann Werner , Quadroni Manfredo , Korostensky Chantal , Gonnet Gaston , Kertesz Michael , James Peter
Published: May 2005

Recently the determination of the genome sequences of three procaryotes (Haemophilus influenzae, Methanococcus jannaschii and Mycoplasma genitalium) as well as the first eucaryotic genome (Saccharomyces cerevisiae) were completed. Between 40–60% of the genes were found to code for proteins to which no function could be assigned. We describe an approach which combines proteome analysis (mapping of expressed proteins isolated by two‐dimensional polyacrylamide gel electrophoresis to the genome) with genetic manipulations to study the complex pattern of protein regulation occurring in Escherichia coli in response to sulfate starvation. We have previously described the upregulation of eight spots on two‐dimensional (2‐D) gels in response to sulfate starvation and the assignment of six of these to entries in the E. coli genome sequence (Quadroni et al., Eur. J. Biochem. 1996, 239, 773–781). Here we describe the identification of the remaining two proteins which are encoded in a sulfate‐controlled operon in the 21.5′ region of the E. coli genome. Upregulated protein spots were cut from multiple 2‐D gels collected and run on a modified funnel gel to concentrate the proteins and remove the sodium dodecyl sulfate before digestion. The peptide masses obtained from the digests were used to search the SwissProt database or a six‐frame translation of the EMBL DNA database using a peptide mass fingerprinting algorithm. A digest can be reanalyzed after deuterium exchange to obtain a second, orthogonal data set to increase the confidence level of protein identification. The digests of the remaining unidentified proteins were used for peptide fragment generation using either post‐source decay in a matrix‐assisted laser desorption ionization (MALDI) time‐of‐flight mass spectrometer or collision‐induced dissociation (CID) coupled mass specrometry (MS/MS) with triple stage quadrupole or ion trap mass spectrometers. The spectra were used as peptide fragment fingerprints to search the SwissProt and EMBL databases.
Specific sample preparation in colorectal cancer
Authors: Reymond Marc A. , Sanchez Jean‐Charles , Schneider Claus , Rohwer Peter , Tortola Silvia , Hohenberger Werner , Kirchner Thomas , Hochstrasser Denis F. , Köckerling Ferdinand
Published: May 2005

Large tissue samples from ten patients operated for colorectal cancer were prepared in the operating room in iced phosphate buffered saline, containing ethylene diaminetetraacetic acid and protease inhibitors. After cutting the specimens into small fragments, the tissues were gently pressed through a steel mesh. Membranes were permeabilized in chilled ethanol 70% to allow cytosolic fluoresceine isothiocyanate labeling, performed with anti‐cytokeratin (CAM 5.2) antibodies. Samples were quantitatively sorted with a fluorescence activated cell sorter (FACS) and denatured before processing separation by two‐dimensional electrophoresis on polyacrylamide gels. This procedure made it possible to sample about 4 × 107 viable normal and tumoral cells before fixation, and up to 4 × 106 cells after FACS. The gels run before and after fixation showed no major differences. The rate of cytokeratin‐positive cells in the samples was the following (mean, Cl 5–95%): mucosa 29.5% (8.9–66.7%), tumor 44.3% (6.6–94.8%). The epithelial cell content in colorectal cancer and normal mucosa shows important intersample variations. This is important for any comparison of fresh samples, whether at DNA, RNA, or at the protein level. We propose a method allowing the preparation of pure epithelial cell samples from normal and tumoral colonic fresh mucosa.
Five cases of forensic short tandem repeat DNA typing
Authors: Schmitt Cornelia , Benecke Mark
Published: May 2005

In medicolegal samples DNA is often broken into fragments. In many cases, only the amplification of short tandem repeated DNA stretches (STRs), which are located in noncoding regions, allows DNA typing of such degraded materials. To demonstrate the high diversity of biological materials which forensic biologists have to deal with, and to outline the success rates and limits of the method, we describe five cases (minute amount of tissue on barrel, tissue in decay, tumor tissue, sperm after multiple rape, stored urine samples) in which forensic DNA typing was successfully performed by use of the short tandem repeats HUMDHFRP2, HUMD8S306, HUMCD4, HUMF13A1, HUMTH01, HUMVWA, and HUMFES

Presented at the “Elektrophorese Forum ”︁96” meeting of the German Electrophoresis Society, held at the Technical University Munich, October 23–25, 1996.

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Resolution of acylated dipeptide stereoisomers by capillary electrophoresis using sulfobutylether derivatized β‐cyclodextrin
Authors: Skanchy David J. , Wilson Richard , Poh Tan , Xie Gaung‐H. , Demarest Charles W. , Stobaugh John F.
Published: May 2005

The separation of enantiomerically and diastereomerically related stereoisomers of acylated Asp‐Phe dipeptides was explored using capillary electrophoresis (CE). This series of dipeptides included the α‐L,L parent compound and the three other potential Asp containing stereoisomers (α‐D,D, α‐L,D, and α‐D,L), as well the four possible isoAsp containing stereoisomers (β‐L,L, β‐D,D, β‐L,D and β‐DL). The separation of these substances was explored using both neutral and charged cyclodextrins as the stereoisomer selector added to the running electrolyte. The major experimental parameters investigated included pH, the cyclodextrin type, and the cyclodextrin concentration. Due to differences in the pKa values of the carboxylic acid groups, adjustment of the separation buffer to between pH 3.0 and 4.0 provided for sufficient electrophoretic mobility differences to result in excellent separations of the diastereomerically related peptides in this pH region. The resolution of the enantiomerically related peptide stereoisomers was accomplished using low concentrations (1 mM) of the anionic cyclodextrin derivative, sulfobutylether‐β‐cyclodextrin (SBE‐β‐CD). This negatively charged cyclodextrin was found to be superior for the resolution of the enantiomerically related peptides as compared to native β‐cyclodextrin or the neutral derivatives, dimethyl β‐cyclodextrin and hydroxypropyl β‐cyclodextrin. An alternative approach using anionic or neutral surfactants in conjunction with the SBE‐β‐CDs was also explored and found to be successful but problematic.