bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural basis of electrophoretic variants of rat α‐fetoprotein
Authors: Ehara Akira , Nishi Shinzo
Published: May 2005

The molecular basis for electrophoretic variations of rat α‐fetoprotein was studied. Stepwise deglycosylations of proteins and radiolabeling of the sugars indicated that the number of such chains per molecule is two and one for the slow and fast variants, respectively.
Luminography – a new, highly sensitive visualization method for electrophoresis
Authors: Schneppenheim Reinhard , Budde Ulrich , Dahlmann Nicolaus , Rautenberg Peter
Published: May 2005

A highly sensitive method for protein visualization following electrophoresis and protein blotting was developed. The method is based on the light‐emitting reaction of luminol and hydrogen peroxide catalyzed by horseradish peroxidase. The luminescent assay can be applied either to the native gel or after protein blotting, and it has a sensitivity two orders of magnitude higher than that achieved with chromogenic detection systems. Analogous to autoradiography the luminescent signal is recorded on an X‐ray film with similar sensitivity. We present several examples of application emphasizing the general versatility of this innovative method.
Oxidation of cysteine to cysteic acid in proteins by peroxyacids, as monitored by immobilized pH gradients
Authors: Chiari Marcella , Ettori Claudia , Righetti Pier Giorgio , Colonna Stefano , Gaggero Nicoletta , Negri Armando
Published: May 2005

It has often been debated whether the presence of persulfate in a polyacrylamide gel could lead to the oxidation of cysteine (Cys) in proteins to cysteic acid. In fact, direct incubation of bovine serum albumin (BSA) with peroxodisulfate and periodate barely alters the isoelectric point (pI) and does not produce any cysteic acid. In contrast, caroate (peroxomonosulfate) and perphathalate strongly lower the pI of BSA. In the former case it as demonstrated that 4‐ Cys (of a total of 35) were converted into cysteic acid. Perphthalate was found to be, by far, the strongest oxidant: 15 (of 35) Cys residues were oxidized to cysteic acid and all methionine groups were destroyed.
Description of a new variant in the gluamateoxaloacetate transaminase system by thin‐layer agarose gel isoelectric focusing
Authors: Farnbacher Oliver , Weidinger Sebastian
Published: May 2005

Thin‐layer isoelectric focusing in agarose within the pH range of 4.0–6.5 has shown a high resolution of the soluble cytoplasmic glutamate‐oxaloacetate transaminase (GOT1) banding pattern. The complex pattern of the common GOT1 phenotype consists of eight bands with different intesities. A genetic variant of GOT1, which has been designated GOT1 Mexico, could tentatively be identified.
Fluctuating bond model of DNA gel electrophoresis
Authors: Schönherr Götz , Noolandi Jaan
Published: May 2005

We present a Monte Carlo algorithm that allows a small length scale numerical study of DNA gel electrophoresis in high electric fields, similar to the fluctuating bond model for dynamical properties of polymeric systems. This approach combines advantages of lattice Monte Carlo method with those from continuous Brownian dynamics algorithms, and also takes into account the persistence length of DNA, as well as the random nature of the gel. The initial orientation and acceleration of a random‐walk DNA conformation shows a number of features that can be related to experimental results. The detailed description of DNA motion provided by this approach may lead to a first realistic computer study of the process of DNA sequencing.
Application of schlieren optics to, real‐time monitoring of protein electrophoresis in crosslinker‐free linear polyacrylamide solution
Authors: Takagi Toshio , Kubota Hidehiro , Oishi Seizo
Published: May 2005

Molecular sieving effect of crosslinker‐free linear polyacrylamide solutions could be visualized in a real‐time mode using schlieren optics for electrophoresis of proteins. Since linear polymer solutions are going to find application in capillary electrophoresis (Zhu, M., et al., J. Chromatogr. 1989, 480, 311–319), the above approach may be useful for obtaining basic data of electrophoresis of proteins and nucleic acids in such media under conditions free from the predominant wall effect pertinent to the use of a capillary.
Detection of gelatinolytic enzyme activities after sodium dodecyl sulfate‐electrophoresis and protein blotting
Authors: Moos Jiri
Published: May 2005

Visualization of proteases with gelatinolytic activity in sodium dodecyl sulfate gels is described. After conventional sodium dodecyl sulfate‐polycrylamide gel electrophoresis, proteins are transferred onto nitrocellulose membranes preincubated with 0.3% gelatine. During the protein electrotransfer, the proteases are renaturated and their enzymatic activity is restored. After nonspecific protein staining, bands with proteolytic activity appear as white areas on a dark background.
Quantification of proteins in sample buffer for sodium dodecyl sulfate‐polyacrylamide gel electrophoresis using colloidal silver
Authors: Dráber Pavel
Published: May 2005

A rapid and simple assay for quantification of proteins in sodium dodecyl sulfate‐sample buffer is described. Proteins are bound to a nitrocellulose membrane, stained with colloidal silver, and quantified by transmission densitometry. The staining requires 1 μL of protein sample and takes less than 20 min. Good linearity between the staining intensity and amount of proteins is in the range of 5–100 ng.
A study of protein synthesis in cells cultured from involved psoriatic skin
Authors: Easty David J. , Patel Ketan , Otto William R. , Dunn Michael J. , Kiil J. , Evans David J.
Published: May 2005

Using histochemical techniques an abnormal programme of epidermal differentiation has been well documented in psoriasis. In order to characterise further the biochemistry of this process we have cultured dermal fibroblasts and epidermal keratinocytes from involved psoriatic skin. This has facilitated metabolic radiolabelling of skin cells and analysis of protein synthesis by two‐dimensional polyacrylamide gel electrophoresis. The expression of keratin and differentiation markers was identical to that of normal keratinocytes, suggesting that psoriatic epidermal differentiation is not truncated in vitro as has been postulated to be the case in vivo. Low molecular mass components (5–8.5 kDa), previously shown to be upregulated in suprabasal keratinocytes, were detected in epidermal fractions from psoriatic skin enriched for basal cells. Of especial interest was a component of 26 kDa, pI 5.9, which was highly upregulated in psoriatic as compared to normal cultured keratinocytes and was not detected in fibroblasts. These findings are in accord with a qualitatively abnormal pattern of differentiation for keratinocytes in the involved psoriatic epidermis.
The application of two‐dimensional polyacrylamide gel electrophoresis to medical microbiology: Molecular epidemiology of viruses and bacteria
Authors: Cash Phillip
Published: May 2005

A variety of molecular methods can be used to identify protein and nucleic acid markers with which to investigate the epidemiology of viruses and bacteria. This paper reviews the application of two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) for studying microbial molecular epidemiology. A small format 2‐D PAGE system is described for locating protein markers in group B coxsackie viruses (CVB) and Haemophilus infuenzae isolates. Representative isolates of CVB serotypes 2, 4, and 5 were compared by analysing the intracellular proteins present in CVB‐infected HEp‐2 cells by 2‐D PAGE protein gels. Although some of the virus‐induced proteins had similar electrophoretic mobilities, the three serotypes could be distinguished from each other on the basis of a major virus induced protein of molecular weight between 39 000 and 43 000. Protein differences were demonstrated among six serotype 2 CVB (CVB‐2) isolates. Four clinical CVB‐2 isolates collected over a period of four months had indistinguishable two‐dimensional protein profiles. Comparison of the two‐dimensional protein profiles of cloned virus stocks prepared from a single clinical CVB isolate demonstrated that it was a heterogeneous virus population. The proteins of nontypable and type‐b H. influenzae isolates were compared. Up to 160 proteins, detected by staining with Coomassie Brilliant Blue R, were resolved by 2‐D PAGE. Although protein differences between individual bacterial isolates were detected, comparable two‐dimensional protein profiles were found for the two groups of H. influenzae isolates. There was no similarity in the two‐dimensional protein profiles of H. influenzae and Aeromonas. Potential protein markers were identified that may be useful in long‐term studies of H. influenzae epidemiology.
Temperature‐dependent spot positional variability in two‐dimensional polypeptide patterns
Authors: Görg Angelika , Postel Wilhelm , Friedrich Claudia , Kuick Rork , Strahler John R. , Hanash Samir M.
Published: May 2005

The effect of temperature, at which isoelectric focusing with immobilized pH gradients is performed, on spot positions and pattern quality in two‐dimensional (2‐D) Electrophoresis was examined. Increased temperatures revealed improved 2‐D patterns with respect to sample entry, resolution, and background staining. Focusing at 20°C was superior to focusing at 10 and 15°C. Even at 30°C, a pattern of well‐resolved polypeptide spots with a minimum amount of horizontal streaking at the basic end was observed. A computer‐based analysis showed that a substantial proportion of polypeptides assumed altered positions in the 2‐D pattern in relation to temperature. Mobility shifts of polypeptides were more variable on the neutral part than on the acidic or basic end. The mobility shifts were not restricted to one direction for all the spots whose migration was altered. However, for any given spot, the direction was the same with subsequent increments of temperature. The results clearly demonstrate that a defined temperature for first‐dimensional isoelectric focusing is a requirement for the reproducibility of 2‐D Electrophoresis. After elimination of the cathodic drift, a major source of variability in 2‐D patterns, associated with carrier ampholytes, temperature control becomes a critical parameter.
Comparison of 14C‐amino acid mixture and [35S]methionine labeling of cellular proteins from mouse fibroblast C3H10T1/2 cells by two‐dimensional gel electrophoresis
Authors: He Chaoying , Merrick B. Alex , Mansfield Betty K. , Hite M. Catherine , Daluge Dennis R. , Selkirk James K.
Published: May 2005

Total Cellular proteins from mouse C3H10T1/2 fibroblasts were compared by two‐dimensional (2‐D) gel Electrophoresis after radiolabeling with [35S]methionine (35S‐Met) or 14C‐amino acids (14C‐AA). 35S‐Met labeling of protein was three to four times greater than 14C‐AA incorporation over a 24 h period. Automated comparative analysis of replicate fluorographs after 6, 12, and 24 h of labeling showed considerable homology between radiolabeling methods. More than 88% percent of 35S‐Met and 14C‐AA‐labeled proteins were common at each time point. However, the total number of 35S‐Met‐labeled proteins dropped from 6 to 24 h while the number of 14C‐AA‐labeled proteins increased. Additionally, twenty‐one proteins were uniquely labeled by 14C‐AA that were not detectable by 35S‐Met over the labeling period. Densimetric analysis showed that several 35S‐Met and 14CAA‐labeled proteins exhibited time‐related diferences in radiolabel incorporation while most proteins remained relatively constant. Protein patterns of silver‐stained gels from 6 to 24 h were highly registered and showed few qualitative differences. Proteins detected in radiolabeled gels were generally, but not always, found in silver‐stained gels. Thus, 35S‐Met appears better suited for short‐term radiolabeling of Cellular protein while more comprehensive labeling of protein occurs with 14C‐AA during prolonged incubation of Cell cultures under present experimental conditions.
High resolution scanning of DNA autoradiographs
Authors: Russell J. R.
Published: May 2005

The sizes of DNA fragments (alleles) on autoradiographs are measured by means of a computerised video scanner developed in this laboratory. This report investigates the advantages of using higher resolution equipment for this purpose. High resolution is simulated by using existing equipment and moving the video camera closer to the autoradiograph. Test autoradiographs, used to validate the original scanner, have been rescanned to determine the degree of improvement that could be obtained using expensive high resolution equipment.
Dodecyl maltoside detergent improves resolution of hepatic membrane proteins in two‐dimensional gels
Authors: Witzmann Frank , Jarnot Bruce , Parker Dona
Published: May 2005

This report describes the incorporation of an alkyl maltoside detergent in two‐dimensional polyacrylamide gel Electrophoresis (2‐D PAGE) sample lysis buffer in order to improve resolution of protein patterns separated by nonequilibrium pH gradient Electrophoresis. Membrane‐associated proteins with alkaline isoelectric points form horizontal streaks on two‐dimensional electrophoretograms when solubilized with conventional nonionic detergent. Dodecyl maltoside enhances protein delipidation during solubilization and improves pattern resolution and protein mobility.
On the computational approach to immobilized pH gradients
Authors: Celentano Fabrizio C. , Gianazza Elisabetta , Righetti Pier Giorgio
Published: May 2005

The unified treatment for computing the pH of complex mixtures of mono‐ and polyprotic buffers, including ampholytes, as utilized in the gradient simulation program PGS, is presented. Its ability to compute pH, buffering power and ionic strength is shown by discussing a few simulations. The problems arising in the automatic formulation of optimal mixtures are presented, as well as the merits and limits of several target functions utilized in such optimizations. It is shown that no universal target function exists and that a proper optimization method should account for the fact that more than one formulation is possible for a given pH range.
Microsequencing of proteins recorded in human two‐dimensional gel protein databases
Authors: Rasmussen Hanne H. , Van Damme Josef , Puype Magda , Gesser Borbala , Celis Julio E. , Vandekerckhove Joël
Published: May 2005

Sixty‐six human proteins recorded in the master transformed human epithelial amnion cells (AMA) (55) and keratinocyte (11) two‐dimensional gel protein databases have been microsequenced since the last publication of the AMA database (Electrophoresis 1990, 12, 989–1071). Coomassie Brilliant Blue stained protein spots cut from several (up to 40) dry gels were concentrated by elution‐concentration gel electrophoresis, electroblotted onto polyvinylidene difluoride membranes and in situ digested with trypsin. The eluting peptides were separated by reversed‐phase high performance liquid chromatography (HPLC), collected individually and sequenced. Computer searches using the FASTA and TFASTA programs from the Genetics Computer Group indicated that 29 of the analyzed polypeptides correspond to hitherto unknown proteins.
A two‐dimensional gel database of rat liver proteins useful in gene regulation and drug effects studies
Authors: Anderson N. Leigh , Esquer‐Blasco Ricardo , Hofmann Jean‐Paul , Anderson Norman G.
Published: May 2005

A standard two‐dimensional (2‐D) protein map of Fischer 344 rat liver (F344MST3) is presented, with a tabular listing of more than 1200 protein species. Sodium dodecyl sulfate (SDS) molecular mass and isoelectric point have been established, based on positions of numerous internal standards. This map has been used to connect and compare hundreds of 2‐D gels of rat liver samples from a variety of studies, and forms the nucleus of an expanding database describing rat liver proteins and their regulation by various drugs and toxic agents. An example of such a study, involving regulation of cholesterol synthesis by cholesterol‐lowering drugs and a high‐cholesterol diet, is presented. Since the map has been obtained with a widely used and highly reproducible 2‐D gel system (the Iso‐Dalt® system), it can be directly related to an expanding body of work in other laboratories.
Simultaneous Ferguson plot analysis, using electrophoresis on a single agarose pore gradient gel, of DNA fragments contained in a mixture
Authors: Gombocz Erich , Chrambach Andreas
Published: May 2005

A recent study [1] has demonstrated the feasibility of obtaining Ferguson plots in agarose gel electrophoresis, using a single pore gradient gel. We now report three remedies for defects in the previous experimental approach: (i) UV‐absorbing media for density stabilization of the gel is avoided by replacing 5‐(N‐2, 3‐dihydroxypropylacetamido)‐2, 4, 6‐triiodo‐N,N′bis (2, 3‐dihydroxypropyl) isophthalamide (Nycodenz) with heavy water; this renders the method applicable to ethidium bromide‐labeled DNA. (ii) The density stabilizing medium is kept from having an effect on field strength. (iii) Data collection by uninterrupted time‐lapse photography is possible by using an apparatus with a quartz window. These three measures make the method practical for the gel electrophoretic identification and physical characterization of DNA species, potentially up to 50 kb in size.
Effective recovery by KCl precipitation of highly diluted muscle proteins solubilized with sodium dodecyl sulfate
Authors: Carraro Ugo , Rizzi Corrado , Sandri Marco
Published: May 2005

A new, improved method is described, which takes advantage of the low solubility of potassium dodecyl sulfate (KDS), to concentrate sodium dodecyl sulfate‐solubilized proteins from nanomolar solutions by KDS precipitation. The method was applied to proteins differing in Mr and pI. The effect of varying KCl, and the pH and/or concentration of the buffer on KDS‐protein precipitation was studied. These parameters may be chosen to allow selective repartition of specific proteins in the pellet or supernatant. After precipitation, the hundred‐ or thousand‐fold concentrated solutes are easily resuspended in small volumes of any required medium. Though initially experimented to recover muscle proteins from effluents of electroendosmotic preparative gel electrophoresis, the method proved to be of general interest as a powerful tool to recover proteins from highly diluted solutions.
Immobilized pH gradients (IPG) simulator–an additional step in pH gradient engineering: I. Linear pH gradients
Authors: Tonani Carlo , Righetti Pier Giorgio
Published: May 2005

A new computer program, called immobilized pH gradients (IPG) simulator, is proposed for calculating and optimizing any recipe for use in isoelectric focusing in immobilized pH gradients. Unlike our previous monoprotic electrolyte gradient simulation (MGS) and polyelectrolyte gradient simulation (PGS) programs, based on minimizing CV(β), the present program has as a target function the minimization of the quadratic moment around zero of the residuals (μ2). With this algorithm it is possible to formulate IPG recipes which have deviations from linearity well below 1% of the given pH interval (a limit set with the previous MGS and PGS programs), in fact, as small as 0.1–0.2% (in pH units). The new simulator performs 2–3 times better than the previous ones in the pH 4–10 range, and is absolutely necessary when working outside this range, at extreme pH values, where CV(β) cannot work against the buffering power of bulk water, thus generating pH recipes with huge deviations from linearity. In the latter cases, μ2 perform 10 times better than CV(β). When utilizing strong titrants for extended pH intervals, the “all or none” rule has been discovered: such titrants should always be used in tandem, since omission of one of the two at either the acidic or basic extremes produces strongly distorted pH profiles. Our new, most powerful simulator also contains equations for creating nonlinear gradients, notably: concave and convex exponentials and sigmoidal (see the companion paper: Righetti, P. G. and Tonani, C., Electrophoresis 1991, 12, 1021–1027)

Program available from Fluka Chemie AG, Buchs, Switzerland

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A genetic factor model for the statistical analysis of multilocus DNA fingerprints
Authors: Krawczak Michael , Bockel Barbara
Published: May 2005

A novel concept is described for the statistical analysis of multilocus DNA fingerprints. Utilizing this method, it is shown by simulation that the application of multilocus DNA fingerprints to paternity testing is robust against deviations from idealistic assumptions made about underlying models and parameters. Partial homozygosity, allelism and linkage at the DNA loci involved, as well as variations in estimates of band‐sharing probabilities were studied for effects on the resulting paternity probabilities. None of the above‐mentioned phenomena appear to change these values to an extent relevant for decision making in paternity cases.
Capillary electrophoretic analysis of flavonoids
Authors: Seitz Ulrike , Oefner Peter J. , Nathakarnkitkool Suraphol , Popp Michael , Bonn Günther K.
Published: May 2005

Combining the effects of electrophoresis and electroendosmosis, flavonoids were separated in less than ten minutes in a fused silica capillary tube with a borate buffer adjusted to pH 10. An increase in the concentration of borate from 0.1 to 0.2 M resulted in longer migration times due to a decrease in electroosmotic flow, but also in improved selectivity and higher resolution of flavonoids. The calibration curve of rutin showed a detection limit of 0.02 mg/mL and linearity over its pharmaceutical concentration range. Using an internal standard of known concentration, the content of rutin in a methanolic extract of Sambuci flos could be determined with a coefficient of variation as small as 3.8% by the molar ratio – peak area ratio method.
cDNA sequence of three cysteine‐rich clusters in the iron‐sulfur subunit of complex II (succinate‐ubiquinone oxidoreductase) from Caenorhabditis elegans determined by automated DNA sequencer
Authors: Kita Kiyoshi , Mizuchi Daisuke , Wang Hua , Takamiya Sinzaburo , Aoki Takashi , Kojima Somei
Published: May 2005

Homology probing by using mixed primers for polymerase chain reaction (PCR) and a subsequent sequence analysis by automated DNA sequencer were applied to determine a partial cDNA sequence of the iron‐sulfur subunit of complex II (succinate‐ubiquinone oxidoreductase). Complex II is a membrane‐bound flavoenzyme, which catalyzes the oxidation of succinate to fumarate in the tricarboxylic acid cycle, and it is a component of the mitochondrial and bacterial respiratory chains. In this study, the partial amino acid sequence of iron‐sulfur subunits in Caenorhabditis elegans mitochondria was deduced from the DNA sequence obtained from cDNA‐PCR. Mixed oligonucleotide primers corresponding to two conserved regions which appear to be the binding site for the prosthetic group were used. The product of PCR was cloned into plasmid vector pUC 119 and the sequence was determined from double strand plasmid DNA by the dideoxy method using of one‐dye, four‐lane type the automated DNA sequencer (DSQ‐1, Shimadzu). The PCR product contained 483 nucleotides and its deduced amino acid sequence was highly homologous with that in human liver (68.9%) and that of Escherichia coli sdh B product (50.3%). As expected, striking sequence conservation was found around the three cysteine‐rich clusters which have been thought to comprise the iron‐sulfur centers of the enzyme.
Electrophoresis and detection of tin‐labeled DNAs on open‐faced gels
Authors: Doktycz Mitchel J. , Arlinghaus Heinrich F. , Allen Robert C. , Jacobson K. Bruce
Published: May 2005

Alternative protocols are necessary for the use of polyacrylamide gel electrophoresis in genome scale sequencing and mapping studies. The use of radioisotopes and manual gel reading will have to be replaced with a flexible labeling system that can be detected at levels similar to or better than radioisotopes but allows automated, high‐speed detection. Labeling with stable isotopes is such an alternative. These nondecaying isotopes have the potential to be detected in sub‐attomole quantities, despite being surrounded by the gel matrix, due to the high selectivity and sensitivity of resonance‐ionization spectroscopy coupled with a mass spectrometer. In this study the detection limits of sputter‐initiated resonance ionization spectroscopy (SIRIS) are investigated using thin, open‐faced polyacrylamide gels supported by plastic. This system allows reproducibility and flexibility in the choice of gel size and buffer system since the gel can be cast, washed free of polymerization by‐products, dried, and stored until use. Various concentrations of an Sn‐labeled oligomer were run on these gels and loads of 5 femtomoles/mm could be detected on a 240 μm thick gel. Gels as thin as 60 μm lower the detectable concentration loads to 1 femtomole/mm. The limiting factor is tin contamination in the gel which, if reduced, will further increase detection. Polymerase chain reaction (PCR) products can also be labeled and detected using Sn isotopes, which could prove useful in mapping studies. Also presented are techniques which will facilitate resolution of these PCR products on open‐faced gels by employing discontinuous buffers systems and DNA mobility modifiers.
Automated magnetic preparation of DNA templates for solid phase sequencing
Authors: Wahlberg Johan , Holmberg Anders , Bergh Staffan , Hultman Thomas , Uhlén Mathias
Published: May 2005

An integrated protocol for solid‐phase DNA sequencing using a robotic work station is described involving magnetic separation of DNA and analysis of the sequencing product by electrophoresis with automated detection of the fluorescently labeled fragments. The method, which is based on magnetic beads in combination with streptavidin‐biotin technology, can be used for sequencing both genomic and plasmid DNA. The DNA template is obtained by the polymerase chain reaction (PCR). Protocols to prepare five and ten immobilized samples is described, giving 10 and 20 single‐stranded templates, respectively. The magnetic purification steps are performed in a microtiter plate and this allows for an integrated scheme involving a subsequent procedure for automated primer annealing and sequencing reactions. Here, the procedure is examplified by direct genomic sequencing of DNA in blood sample from a human immunodeficiency virus (HIV)‐ infected patient and a cloned human antibody DNA fragment using fluorescently labeled sequencing primers.
High‐sensitive fluorescent DNA sequencing and its application for detection and mass‐screening of point mutations
Authors: Hattori Masahira , Yoshioka Katsuji , Sakaki Yoshiyuki
Published: May 2005

We describe a rapid and sensitive DNA sequencing method for an automated fluorescent DNA sequencer (AFDS) and its application for detection of point mutations. The method is based on an improved cycle sequencing procedure in which only 10–50 fmol of template DNA is required. Furthermore, it is able to use crude DNA preparation as a template as well as the purified one. Thus, the improved method provided a simplified procedure for sequencing of various types of DNA, including cosmid DNA, in which purification steps were unnecessary. We also developed a novel system for detection of point mutations using AFDS. A set of four lanes is used for the parallel analysis of single‐base profiles of four different samples, instead of for the four‐base profile of a sample. The AFDS exhibits the base profiles of the samples with four different colors in the analyzed data, which enables us to identify a mutation as an additional peak with a color specific for the lane. The feasibility of our system was tested by analyzing polymerase chain reaction (PCR)‐amplified genomic DNAs from four individuals including a carrier of a mutation of C to T. The mutation was clearly identified as an additional “T” peak of a color specific for the carrier. The mutation was also detectable even if 16 individuals including the carrier were simultaneously analyzed on a set of four lanes (four individual samples for each lane). Thus, the novel system is useful for simultaneous detection of mutations in a large number of individual samples.
Manual sequencing using pulsed field
Authors: Turmel Chantal , Brassard Eric , Noolandi Jaan
Published: May 2005

The use of pulsed fields in manual sequencing opens up the compression zone found with a DC field and extends the range of resolution from a few hundred bases to several thousand bases. The band inversion problem is overcome with the proper pulsing conditions, and the bands are sharper than for the DC field case. Accurate visual reading is possible up to about 800–900 bases. The method is compatible with automation techniques, since the band spectrum is stretched continuously during migration, and the smaller fragments are run off the gel.
A PacI/SwaI map of the Pseudomonas aeruginosa PAO chromosome
Authors: Römling Ute , Tümmler Burkhard
Published: May 2005

The PacI/SwaI macrorestriction map of the 5.9 Mb Pseudomonas aeruginosa PAO (DSM 1707) genome was constructed by fragment size comparisons of complete single and double digests and hybridization with probes of known genomic localization. P. aeruginosa PAO contains five recognition sites for PacI and six sites for SwaI. An integrated DpnI/PacI/SpeI/SwaI map was obtained by fragment pattern and Southern blot hybridization analyses of all combinations of double digestions and by two‐dimensional pulsed‐field gel electrophoresis mapping protocols.
Identification of peaks in capillary zone electrophoresis based on actual mobilities
Authors: Vespalec Radim , Gebauer Petr , Boček Petr
Published: May 2005

A procedure is proposed for the calculation of the actual effective mobility of a zone from its migration time. It is based on the use of internal standards with known mobilities; the use of two internal standards provides reliable mobility data even if the magnitudes of the effects of sample composition, capillary temperature, capillary length, migration distance, used voltage, as well as the tube length occupied by the injected sample are unknown. Formulas have been derived for the calculation of the actual mobilities, and their experimental verification has been carried out by using a model set of anionic solutes with mobilities ranging from −56 to −20 × 10−9 m2V−1s−1 and chloride as the ion modelling the effect of the sample matrix.
Atrazine and simazine determination in river water samples by micellar electrokinetic capillary chromatography
Authors: Desiderio Claudia , Fanali Salvatore
Published: May 2005

A rapid and sensitive micellar electrokinetic capillary chromatography analytical method was used for the determination of chlorotriazine herbicides in river water samples. Several electrolyte systems in the pH range 7–10 were tested in order to optimize the separation. The two compounds were separated in less than 10 min and the determination limit was about 0.4 ppb for each herbicide. Recovery values of the method were in the range 80%–117%.
Applications of an automated apparatus for two‐dimensional electrophoresis, Model TEP‐1, for microsequence analyses of proteins
Authors: Nokihara Kiyoshi , Morita Naoki , Kuriki Tomoko
Published: May 2005

Two‐dimensional electrophoresis has become a rapid and powerful separation method for the preparation of samples for sequence analysis depending on the development of automated microsequencers accompanied by a blotting technique. We have constructed an automated apparatus for two‐dimensional electrophoresis, Model TEP‐1, which allows precise analytical runs under computercontrol with a fully automated transfer from the first‐ to the second‐dimensional gel. In the present paper we describe several applications of TEP‐1 followed by blotting and sequencing to reveal the primary structure of proteins in a complex mixture. Several known and unknown proteins were analyzed. Enzymatic digests of proteins could be directly applied onto the TEP‐1, in which a gel of lower molecular weight proteins was used as the second dimension. Subsequent blotting and sequencing provided internal sequence information. The use of TEP‐1, followed by blotting, provides spots containing ca. 10–100 pmol protein. These figures permitted the identification of 10–40 amino acid residues from the N‐terminus. The TEP‐1 provides a simple and rapid separation of complex mixtures obtained from natural sources and chemical, biological or genetic products.
Equipment for rapid homogenization of high numbers of plant tissue for electrophoretic analysis of proteins
Authors: van den Berg Bartel M. , Burg Hendrik C. J. , Tamboer Jan H. A. , Grapendaal Bob
Published: May 2005

A homogenizing system was developed to process multiple samples of various plant tissues. The equipment consists of a motor‐driven stainless steel homogenizer that fits perfectly into a hole of a disposable plate. Hard plant material like melon or squash seeds (maximal length of 1.4 cm) could be homogenized within 10 s by forcing the homogenizer towards the bottom of a 24‐ or 48‐well tissue culture plate. Smaller seeds could be homogenized within 10 s in a similar way using a 96‐well tissue culture plate. Leaf tissue, or other types of soft plant tissue, could be homogenized with the same equipment. For homogenization of small samples, e.g. pistils of small flowers, a 60‐well Terasaki plate was used. For homogenization of several hundreds of leaf tissue samples, an apparatus was developed that contains 12 homogenizers driven simultaneously by an electromotor. A tissue culture plate containing 96 leaf samples could be handled in this way in less than 10 min. The performance of the equipment was evaluated by analyzing homogenates of several types of plant tissue by different electrophoretic techniques.
An explanation of the achromatic bands produced by peroxidase isozymes in polyacrylamide electrophoresis gels stained for malate dehydrogenase
Authors: Fieldes Mary Ann
Published: May 2005

When plant tissue extracts are electrophoresed on polyacrylamide gels and the gels are stained for malate dehydrogenase by the standard NAD‐dependent dehydrogenase reaction, terminating in the formation of reduced Nitroblue Tetrazolium (NBT), achromatic bands, in addition to the expected chromatic bands, are observed. The achromatic bands are seen when the staining conditions favor a generalized background staining of the gel and have been shown, in a previous study, to be caused by peroxidase isozymes [1]. The present study examined the mechanism by which peroxidase produced the achromatic bands using horseradish peroxidase (HRP). The generalized background staining resulted from the phenazine methosulfate (PMS)‐mediated reduction of NBT. This reduction was enhanced by H2O2 and suppressed by HRP. Peroxidase apparently catalyzes the peroxidative oxidation of reduced PMS, which suppresses the generalized reduction of NBT in gel regions containing peroxidase isozymes producing the achromatic bands. In contrast, however, HRP also appears to catalyze the peroxidative oxidation of reduced NAD, but this reaction increases the reduction of NBT. The results are discussed in the context of the mechanisms proposed by others for the PMS‐mediated reduction of NBT and for the peroxidase‐catalyzed NADH‐dependent formation of H2O2. This peroxidase‐catalyzed reaction has been proposed for the plant peroxidases involved in lignification.
Effects of bile salt exposure on pancreatic duct barrier function and protein release determined by two‐dimensional electrophoresis and lectin‐affinoblotting
Authors: Hänsler Marion , Arendt Thomas , Lange Kay
Published: May 2005

We analyzed effects of a bile salt on pancreatic duct barrier function and protein release into pancreatic juice in cats, using two‐dimensional‐electrophoresis and lectin‐affinoblotting. Preanalyzed pancreatic juice was perfused along the pancreatic duct following duct exposure to a bile salt. In this case, pancreatic duct mucosa showed a partial loss of barrier function to the leakage of exocrine pancreatic proteins dependent upon molecular weight and charge. The release of a sialic acid possessing protein into pancreatic juice was observed.
The use of radioactive bacteriophage proteins as X–Y markers for silver stained two‐dimensional electrophoresis gels and quantification of the patterns
Authors: Gersten Douglas M. , Ramagli Louis S. , Johnston Dennis A. , Rodriguez Lewis V.
Published: May 2005

We have demonstrated the feasibility of using bacteriophage ghost proteins, tritiated by metabolic labeling, as a set of standard markers for two‐dimensional gels in which the proteins are to be detected by silver staining. The results indicate that a 2.5 μg load of phage proteins yields a reproducible silver pattern of 48 spots. The spots can also be readily identified by radioautography and radiofluorography, establishing their value as a standard constellation of markers. Quantification of these patterns by computerized densitometry indicates a general agreement between detection by silver staining and detection by radiofluorography.
Wheat gliadin: Digital imaging and database construction using a 4‐band reference system of agarose isoelectric focusing patterns
Authors: Black Jeffrey A. , Waggamon Kimberly A.
Published: May 2005

An isoelectric focusing method using thin‐layer agarose gel has been developed for wheat gliadin. Using flat‐bed units with a third electrode, up to 72 samples per gel may be analyzed. Advantages over traditional acid polyacrylamide gel electrophoresis methodology include: faster run times, nontoxic media, and greater sample capacity. The method is suitable for fingerprinting or purity testing of wheat varieties. Using digital images captured by a flat‐bed scanner, a 4‐band reference system using isoelectric points was devised. Software enables separated bands to be assigned pI values based upon reference tracks. Precision of assigned isoelectric points is shown to be on the order of 0.02 pH units. Captured images may be stored in a computer database and compared to unknown patterns to enable an identification. Parameters for a match with a stored pattern may be adjusted for pI interval required for a match, and number of best matches.
On the use of dimension‐less parameters in acid‐base theory. I. The buffer capacity of simple ampholyte solutions
Authors: Rilbe Harry
Published: May 2005

A complete mathematical account of the buffer capacity of bivalent ampholytes is given. The consistent use of dimensionless parameters makes it possible to obtain very neat expressions and equations. Direct proof of the well‐known condition K1 ⩾ 4 K2 is presented. Since water is also an ampholyte, its buffer capacity is considered as well.
Polyhydroxy and polyethyleneglycol (meth)acrylate polymers: Physical properties and general studies for their use as electrophoresis matrices
Authors: Zewert Thomas , Harrington Michael
Published: May 2005

A new series of materials have been tested for their suitability as electrophoresis matrices. The mechanical and optical properties of gels composed of polyethyleneglycol (meth)acrylate esters or polyhydroxy (meth)acrylate esters in water and in various concentrations of organic solvents are described. Several crosslinkers including polyethyleneglycol and polyhydroxy di(meth)acrylates, piperazine diacrylate, and bisacrylamide were used in these studies. Electrophoretic migration and separation of a series of protein standards through polyethyleneglycol methacrylate (PEGM) 200, PEGM 400, and glyceryl methacrylate is demonstrated. Further, copolymerization of all of the monomers with acrylamide was performed and the distribution of monomer incorporation into the polymer network calculated. All monomers and copolymers that were examined by IR spectroscopy showed greater than 99% polymerization. These results justify their further study for biomolecule separations.
Identification of Gram‐negative bacteria by isoelectric focusing of soluble cellular proteins
Authors: Margull Andreas , Süssmuth Roland , Schulz Peter
Published: May 2005

Strains of Escherichia coli. Pseudomonas aeruginosa and Proteus mirabilis, isolated from clinical specimens were disrupted by repeated freezing in liquid nitrogen and thawing at room temperature. The samples were separated by isoelectric focusing using polyacrylamide gels. The resulting protein patterns showed clear differences between the three species and made identification possible.
A rapid periodic acid‐Schiff staining procedure for the detection of glycoproteins using the phastsystem
Authors: Van‐Seuningen Isabelle , Davril Monique
Published: May 2005

A procedure for the analysis of glycoproteins and glycopeptides using the PhastSystem with detection by the periodic acid‐Schiff stain is described. Following sodium dodecyl sulfate or nondenaturing polyacrylamide gel electrophoresis and also isoelectric focusing, samples are stained directly for the presence of carbohydrates. By using the PhastSystem, the method is rapid, sensitive, reliable and allows storage of the gels without a change in the stain. As little as 0.1 μg of protein‐associated carbohydrates can be detected. The staining procedure is also used following isoelectric focusing of mucin‐derived glycopeptides to visualize their charge differences and the increase of their isoelectric point after neuraminidase treatment.
The local reciprocal method for sizing DNA fragments: An obscure limitation
Authors: Catterick Tim
Published: May 2005

The local reciprocal method, for sizing DNA fragments, has been found to fail when the experimental data produces a linear relationship. A simple solution for computerised systems is to test for this unexpected condition.
Transverse pore gradient gel electrophoresis of lipoprotein [a] using methylenebisacrylamide gradients
Authors: Smejkal Gary B. , Hoff Henry F.
Published: May 2005

We describe a modification of transverse pore gradient gel electrophoresis in which pore size is regulated by crosslinker proportion (%C) rather than by total monomer concentration (%T). We electrophoresed plasma lipoprotein [a] transversely across linear N,N′‐methylenebisacrylamide gradients and measured mobility as a function of %C. This method allows for the simultaneous assessment of pore sizes generated over a wide range of crosslinker proportions.
Low fluorescence background electroblotting membrane for DNA sequencing
Authors: Chu Tun‐Jen , Caldwell Karin D. , Weiss Robert B. , Gesteland Raymond F. , Pitt William G.
Published: May 2005

A low fluorescence background polypropylene (PP) membrane has been developed for ultimate use as an electroblotting membrane in DNA sequencing based on fluorescence detection. The DNA binding capacity of this membrane is improved by a surface modification using radio frequency plasma discharge (RFPD) in ammonia gas. The RFPD operational parameters are evaluated both in terms of membrane nitrogen content and in terms of the product's capacity for binding radioisotope‐labeled DNA fragments. The surface morphologies of the derivatized membranes are examined by scanning electron microscopy; their mechanical and electrical properties, which are important for the subsequent sequencing procedures, are likewise established. Due to the goal of developing a membrane suitable for multiplex processing, in which the electroblotted DNA must withstand dozens of hybridization/stripping cycles, special attention is given the covalent attachment of DNA to the membrane. The modified PP membrane is evaluated in a multiplex sequencing application using radioisotope‐labeled DNA probes, and found to yield somewhat better binding of a given amount of electroblotted DNA than the commonly used GeneScreen membrane. A tenfold repetition of the probing indicates little loss of signal; the membrane‐bound DNA is stable upon storage and shows no detectable loss in probing efficiency after one month.
Oligonucleotide fingerprinting of plant and fungal genomes: A comparison of radioactive, colorigenic and chemiluminescent detection methods
Authors: Bierwerth Susan , Kahl Günter , Weigand Franz , Weising Kurt
Published: May 2005

Digoxigenated oligonucleotide probes complementary to simple repetitive DNA sequences were introduced into nonradioactive fingerprint analysis of plant and fungal DNA. The fragment patterns, obtained by blot hybridization of TaqI‐restricted DNA from chickpea (Cicer arietinum) and its fungal pathogen Ascochyta rabiei with digoxigenated probes and either a colorigenic or a chemiluminescent detection method, were compared to those obtained with 32P‐labeled probes. In combination with alkaline phosphatase and its chemiluminescent substrate 3‐(2′‐spiroadamantane)‐4‐methoxy‐4‐(3″‐phosphoryloxy)phenyl‐1,2‐dioxetane (AMPPD) digoxigenated oligonucleotides yielded clear‐cut fingerprints with high signal‐to‐background ratios within several minutes of exposure to X‐ray films. The chemiluminescence reaction remained stable for at least two weeks. A comparison of banding patterns obtained by radioactive versus digoxigenin‐based hybridization and detection techniques revealed substantial differences in the relative signal intensities of bands. Both nonradioactive techniques show a tendency to “equalize” band intensity differences. Whereas 32P‐labeled oligonucleotides are also applicable to in situ hybridization with DNA immobilized in dried agarose gels, gel hybridization did not work efficiently with digoxigenated probes and either substrate.
Impact of separation capacity on the isotachophoretic analysis of organic acids in human seminal plasma and prostatic fluid
Authors: Oefner Peter J. , Häfele Robert , Eberle Johannes
Published: May 2005

In the present study, the impact of pH of both the leading electrolyte and the sample on the separation capacity of an isotachophoretic system, used for the analysis of organic acids in human seminal plasma and prostatic fluid, was assessed. Generally, maximum separable amounts of eleven equimolar pairs of anions decreased within a pH range of 2.90–4.25 of the leading electrolyte due to increasing net mobilities of the analytes with rising pH. The values obtained under equimolar conditions were also valid for nonequimolar mixtures as long as the buffer capacity of the leading electrolyte was sufficient to maintain constant mobilities of the ionic species to be separated throughout the capillary. However, when the sample injected has a buffer capacity of its own and a pH higher than that of the leading electrolyte, separation capacity will vary. For that reason, the maximum separable amount of an aqueous solution of phosphate and citrate was higher than that obtained for seminal plasma. Hence, the injection volume had to be reduced in order to ensure complete resolution. Besides phosphate and citrate, which was the prime component of prostatic fluid, lactate, aspartate and glutamate could be quantitatively determined in seminal plasma with a precision of ± 1.5%.