bluuebunny/crossref_metadata_2025_split huggingface dataset:
Detection of gelatinolytic enzyme activities after sodium dodecyl sulfate‐electrophoresis and protein blotting
Authors: Moos Jiri 
Published: May 2005

AbstractVisualization 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

AbstractA 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

AbstractUsing 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

AbstractA 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

AbstractThe 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

AbstractTotal 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

AbstractThe 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

AbstractThis 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

AbstractThe 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

AbstractSixty‐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

AbstractA 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

AbstractA 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

AbstractA 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

AbstractA 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

AbstractA 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

AbstractCombining 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

AbstractHomology 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

AbstractAlternative 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

AbstractAn 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.