bluuebunny/crossref_metadata_2025_split huggingface dataset:
Detection of polypeptides associated with the cold acclimation process in spinach
Authors: Guy Charles L. , Haskell Dale 
Published: May 2005

AbstractExposure of spinach (Spinacia oleracea) seedlings to 5 °C for several days has previously been shown to induce a greater tolerance to the stresses of extracellular freezing. Associated with this response to low temperature, termed cold acclimation, was a subtle shift in protein synthesis and altered polypeptide composition. Two‐dimensional gel electrophoresis was used to study the changes in spinach leaf tissue protein synthesis in an effort to identify polypeptides that may play a central role in the induction of greater freezing tolerance. Through a combination of silver staining, in vivo labeling, and in vitro translation of mRNAs, we identified several high molecular weight polypeptides whose synthesis and presence in spinach leaf tissue were highly correlated with freezing tolerance. Synthesis of these polypeptides was elevated or induced during cold acclimation when freezing tolerance increases, but was rapidly reduced or halted during deacclimation when freezing tolerance declines. The close association of the synthesis of these polypeptides with the induction and loss of freezing tolerance suggested that they could play a role in cold tolerance mechanisms of spinach.
Determination of molecular weight of native proteins by polyacrylamide gradient gel electrophoresis
Authors: Nishizawa Hideyuki , Kita Natsumi , Okimura Sakiko , Takao Emi , Abe Yoshihiro 
Published: May 2005

AbstractAn improved method for the estimation of molecular weights of native proteins by polyacrylamide gel electrophoresis, in 9 cm × 9 cm × 0.05 mm 4–20% T fabric reinforced gradient gels, is described. Plotting the logarithm of the relative mobilities of proteins versus gel concentrations produces lines whose slopes are related to molecular weights.
High resolution of human lactate dehydrogenase: New multiple forms and potential tumor markers
Authors: Romero‐Saravia Oscar , Solem Eivind , Lorenz Matthias 
Published: May 2005

AbstractHuman lactate dehydrogenase (LDH) was investigated by ultrathin‐layer polyacrylamide gel isoelectric focusing (IEF). In serum, approximately 15 bands and in lyzates of erythrocytes approximately 20 bands were detected. Known LDH isoenzymes (identified by markers) appeared in the zymograms as follows: LDH‐1 as a single or double band, LDH‐2 as a single band in serum and in the marker, and as a double band in hemolyzate, LDH‐3 as a double band, and LDH‐4 and LDH‐5 each as a single band. LDH‐1 was partly inactivated, probably due to deamidation in the acidic range of the pH gradient. Potential LDH tumor markers were detected in different tumor cytosols.
Determination of oxalate in human serum by multicolumn isotachophoresis
Authors: Dolník Vladislav , Deml Mirko , Boček Petr 
Published: May 2005

AbstractThe practical usefulness of multicolumn isotachophoresis was demonstrated by the determination of oxalate levels in human serum. 10 mmol/L HC1 and 100 mmol/L Na2HPO4 served as the leading and terminating electrolyte, respectively. For the stabilization of the isotachophoretic zones in large cross section channels a suspension of Bio‐Gel P‐300 in the leading electrolyte was used. For the analysis ca. 1 mL of fresh serum was required and 1.2 mL of 1:1 mixture of serum and the suspension of Bio‐Gel P‐300 in deionized water was applied as sample. The time of analysis ranged between 45–49 min. The detection limit of analysis was determined to be 50 nmol/L. Reproducibility of analysis of 1 μmol/L oxalate in water standard was found to be 2.6% (n = 6).
A system for automated DNA electrophoresis, molecular hybridization and electronic detection: II. Electronic detection
Authors: Zapolski Edward J. , Gersten Douglas M. 
Published: May 2005

AbstractWe have designed and constructed an automated, computer‐controlled, nucleic acid hybridization analysis system (Electrophoresis 1987, 8, 255–261). The system performs 9 simultaneous experiments, beginning with submarine electrophoretic separation of the restriction fragments and including microwave fixation of the separated fragments, denaturation, neutralization, prehybridization, hybridization, washing and drying. The final step is electronic detection of the hybridization pattern. The detector system consists of 90 Geiger‐Mueller detectors arranged to simultaneously sample the 9 hybridizations at 10 positions each. The hybridization matrix is moved across the detectors by a robot arm in increments preprogrammed by the operator and the entire length of the matrix can be counted. The results are printed out as a plot of radioactive counts vs. distance from the origin of electrophoresis. We describe here the characteristics of the detection system.
A method for recovery of native, clonally‐restricted immunoglobulins from agarose gels
Authors: Janson Robert W. , Vertosick Frank T. , Kelly Robert H. 
Published: May 2005

AbstractMultiple low level, clonally‐restricted, immunoglobulins (Ig) are commonly encountered on routine serum protein electrophoresis by clinical laboratories using high resolution zone electrophoresis on agarose. We sought a method for recovering the clonally‐restricted Ig, in native configuration, from clinical laboratory gels as a first step in the investigation of its clinical significance. We found that a two‐stage electrophoretic procedure gave consistently good recoveries. After routine agarose gel electrophoresis, portions of the electropherogram, containing clonally‐restricted Ig, were excised and subjected to flatbed isoelectric focusing in agarose to enhance separation of the individual antibody clonotypes. Multiple slabs, containing the same clonally‐restricted Ig, could be cut from adjacent tracks (i. e., tracks loaded with the same specimen) on the zone electropherogram and applied to a single track on the focusing gel to improve separation and increase yields. The focused gels were cut to isolate slabs containing individual clonotypes. These slabs were washed to remove carrier ampholytes and held at −20 °C overnight. Ig was extracted from the thawed gels, with 61–68 % recovery, by ultracentrifugation following physical disruption of the gel. Antigen binding activity of the recovered Ig was verified by rate nephelometry. Clonally‐restricted antibodies were successfully isolated from an immune animal serum by this procedure and biotinylated for use as probes on Western blots.
Determination of carboxymethyl‐D‐glucoses in the product of hydrolytic depolymerization of carboxymethylcellulose by isotachophoresis
Authors: Pospíšílik Karel 
Published: May 2005

AbstractOptimal conditions for isotachophoretic separation of carboxymethyl‐D‐glucoses formed by acidic depolymerization of carboxymethylcellulose were found. 6‐O‐car‐boxymethyl‐D‐glucose, 2‐O‐carboxymethyl‐D‐glucose and 3‐O‐carboxymethyl‐D‐glucose were identified and determined in the reaction mixture after carboxy‐methylcellulose hydrolysis. Relative reactivity of hydroxy groups in the glucopyranose unit of cellulose decreased in the following order: O(6)H>O(2)H> >O(3)H. This was found to be in agreement with the data published by other authors.
Application of an affinity electrophoretic and in situ oxidation method to the study of the equine protease inhibitory proteins
Authors: Patterson Scott D. , Bell Kevin 
Published: May 2005

AbstractAn affinity method was developed to investigate the interaction between protease and protease inhibitor by incorporating a protease incubation step into a two‐dimensional electrophoretic separation of the plasma protease inhibitory proteins. This involved the application of the isoelectric focusing gel to filter paper saturated in the protease of choice before being placed on the second‐dimensional polyacrylamide electrophoresis gel. General protein staining or immunoblotting was used to detect the protein or ligand in the complex. An in situ oxidation method was developed using the reagent ligand in the complex. An in situ oxidation method was developed using the reagent chloramine T to investigate the effect of this reagent on the complexing abilities and inhibitory activities of the protease inhibitory proteins. Oxidation was performed either after electrophoresis prior to staining for enzyme inhibition or during two‐dimensional electrophoresis prior to the aforementioned protease incubation. The latter allowed the effect of oxidation on complex formation to be examined. Whole plasmas were utilized as the sources of protease inhibitory proteins with the human and mouse being used as models. The equine protease inhibitory system was examined by the two methods and shown to consist of three classes of inhibitory proteins based on their susceptibilities to oxidation and abilities to form complexes with various proteases.
Genetic studies of low‐abundance human plasma proteins: X. Coagulation factor XIIIB variants in blacks
Authors: Kamboh Mohammad I. , Ferrell Robert E. 
Published: May 2005

AbstractHuman coagulation factor XIIIV (F XIIIB) demonstrates genetically determined‐structural variation with three common and several rare alleles. Population genetics studies reveal enormous intra and interracial group variation. In the present study, using isoelectric focusing and immunoblotting, we have determined for the first time the polymorphic occurrence of F XIIIB allelic forms in a native African population, namely Nigerian Blacks. In addition, F XIIIB data have been extended to various US Black populations. The characteristic feature of the black gene pool is the relative high frequency of the F XIIIB*2 allele, the highest being in Nigerians (0.723). The F XIIIB*6 allele is present at a polymorphic level in both the US and Nigerian Blacks and appears to be a unique black allele marker. The present technique has demontrated several new alleles designated: F XIIIB* 18, F XIIIB*22, F XIIIB*23 and F XIIIB*24. Among these new alleles the F XIIIB*23 exists at polymorphic level in both the US and Nigerian Blacks and is another unique Black allele marker of potential significance in population genetics studies.
Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis without a stacking gel: Use for high sample throughput
Authors: Marshall Thomas , Gaston S. J. Stephen , Williams Katherine M. 
Published: May 2005

AbstractSodium dodecyl sulfate‐polyacrylamide gel electrophoresis without a stacking gel minimizes lateral spreading of protein when samples are applied in agarose wells and allows high sample throughput (6 samples/cm gel width). The method is simple and convenient to use and gives comparable resolution to the standard method with 4–20% or 6–30% polyacrylamide gradient gels. Best results are obtained when the upper zone of the separating gel is of low polyacrylamide concentration. This indicates a need for the molten agarose to penetrate and anneal with the separating gel.
Toward a steady‐state pore limit electrophoresis dimension for native proteins in two‐dimensional polyacrylamide gel electrophoresis
Authors: Fawcett John S. , Sullivan James V. , Chrambach Andreas 
Published: May 2005

AbstractPolyacrylamide gel electrophoresis in linear pore gradients (4.8 to 48 %T, 5 %CBis) provides for migration arrest, in a practical sense, after about 5000 Vh for proteins of 290 and 450 kDa, but not for smaller proteins over 20 000 Vh. The arrest is not due to inadequate field strength nor is it caused by water redistribution within pore gradien gels. The possibility is being discussed that exponential pore gradients, and a higher or a lower degree of crosslinking suggested by the literature may be remedies for the present failure to arrest the migration of smaller proteins.
Program in BASIC for ferguson plot analysis, using a personal computer: Application to gel electrophoresis in a continuous buffer
Authors: Gombocz Erich , Chrambach Andreas 
Published: May 2005

AbstractA program in BASIC suitable for personal computers is described which is applicable to gel electrophoresis conducted in a single (continuous) buffer. The curve fitting is to a polynomial function, allowing for an objective selection of the most appropriate curve type and order – linear, convex or concave – in the particular application. Results do not differ significantly from previous programs for evaluation of linear Ferguson plots or of curve fitting to an exponential function for evaluating convex plots, executed on mainframe computers such as the DEC‐10 (Digital) and IBM 370 computers. Thus, the program combines original versatility with, for the first time, the possibility for widespread application of Ferguson plot analysis on personal computers.
Vertical remission densitometry for the evaluation of nontransparent samples
Authors: Taube Dieter , Neuhoff Volker 
Published: May 2005

AbstractGels dried onto filter paper or their blots on membranes are not suitable for densitometric evaluations in the transmission mode because these samples are nearly nontransparent. By scanning in the remission mode, evaluation with good results is possible. Data acquisition by a computer‐controlled high speed scanning photometer and digital image processing affords a spatial resolution and accuracy similar to that in the transmission mode. The signal‐to‐background ratio decreases by 10–30% depending on the smoothness of the support.
Mirror densitometry for higher sensitivity and resolution
Authors: Taube Dieter , Neuhoff Volker 
Published: May 2005

AbstractIn order to increase the pathway of the light inside a gel (or autoradiogram) during scanning, it is placed on top of a mirror and the densitometry is performed in a vertical reflection mode. As the light passes the gel a second time after being reflected by the mirror, the absorbance is nearly doubled. This increase in absorbance results in higher sensitivity and resolution.
Interactions between lipopolysaccharide and outer membrane proteins of Acinetobacter calcoaceticus studied by an affinity electrophoresis system
Authors: Borneleit Petra , Blechschmidt Bernd , Kleber Hans‐Peter 
Published: May 2005

AbstractR‐Form lipopolysaccharides of Acinetobacter calcoaceticus could be incorporated into polyacrylamide gels in an immobile form by adding it directly to the acrylamide‐N, N′‐methylenebisacrylamide polymerization mixture. The separation of A. calcoaceticus 69 V outer membrane proteins in these affinity gels demonstrated a specific interaction with the lipopolysaccharide ligand for one of the proteins. This protein is heat‐modifiable and has an Mr of about 18 000. By incorporation of varying concentrations of lipopolysaccharide, a dissociation constant of the protein‐lipopolysaccharide complex of 0.5 mM could be determined. In comparison, for another A. calcoaceticus strain, CCM 5593, a higher dissociation constant (1.0 mM) – indicative of lower affinity – was obtained.
Sieving of ionic constituents across moving boundaries in gel electrophoresis
Authors: Orbán László , Fawcett John S. , Tietz Dietmar , Chrambach Andreas 
Published: May 2005

AbstractThe representative β‐hydroxyethylmorpholinium‐chloride‐bicinate moving boundary with a trailing ion net mobility relative to Na+ of 0.41, detected by precipitation of chloride with silver nitrate, exhibits a decreasing chloride mobility at increasing polyacrylamide gel concentrations from 3.5 to 45 %T, 5 %CBis. This decrease, largely due to an increase of field strength at constant current, is described by a convex plot of log (mobility) vs. %T (Ferguson plot) and signifies that chloride/bicinate are sieved by the gel. In agarose gels, the same plot of mobility vs. gel concentration is constant below 7 % gel concentration, since in those gels field strength and migration rate remain the same within that gel concentration range. Both in polyacrylamide and in agarose gels the displacement rate of the chloride‐bicinate boundary as a function of the time of electrophoresis or distance migrated remains invariant within 15 %. The plot of log(mobility) vs. gel concentration extrapolated to 0 %T is 5.85 and 5.41 (10−5 cm2 s−1V−1) for polyacrylamide and for agarose (SeaKem HGT‐P, FMC) gels, respectively. The slightly decreased mobility intercept at 0 %T for agarose is presumably due either to the electroendosmotic properties of agarose HGT‐P and/or failure to Sufficiently take into account the flattening of the Ferguson plot in the polyacrylamide concentration range below 3 % in which a transition from a gel to a fluid (sol) medium takes place.
Preparation of plant DNA for separation by pulsed field gel electrophoresis
Authors: Devos Katrien M. , Vercruysse‐Dewitte Daisy 
Published: May 2005

AbstractA method was developed for the preparation of completely intact plant DNA embedded in agarose, and suitable for restriction enzyme digestion. Digestion with restriction enzyme was carried out according to modified protocols of Anand [1] and Kenwrick et al. [2]. The new method of DNA isolation allows the separation of high molecular weight plant DNA by pulsed field gel electrophoresis.
Electrophoresis of human salivary proteins: Response to natural stimulation
Authors: Marshall Thomas , Lynass Rachel C. , Williams Katherine M. 
Published: May 2005

AbstractHuman saliva was analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and the response to different natural stimuli investigated. Serva Blue R revealed pink/violet‐staining proteins (“lumicarmines”). These intensified following stimulation but in an unpredictable and sometimes disproportionate manner, causing pattern changes. This variable response undermines their potential forensic value for suspect identification.
Pulsed field electrophoresis in contour‐clamped homogeneous electric fields for the resolution of DNA by size or topology
Authors: Chu Gilbert 
Published: May 2005

AbstractThe electrophoretic separation of DNA molecules can be controlled by the use of contour‐clamped homogeneous electric fields (CHEF). This paper describes an improved CHEF apparatus with negligible distortion in the electric fields. When the electric field was periodically reoriented, DNA molecules up to 2 megabases were resolved in a highly uniform manner. Furthermore, when the field strength was changed with orientation, topological variations of conventional‐sized DNA molecules were resolved.
Use of the hydroxyl radical and gel electrophoresis to study DNA structure
Authors: Shafer Gwen E. , Price Mary A. , Tullius Thomas D. 
Published: May 2005

AbstractThe hydroxyl radical has been used as a chemical probe to study in solution the structure of DNA and DNA‐protein complexes. The hydroxyl radical abstracts a deoxyribose hydrogen atom, cleaving one strand of the DNA. The cutting pattern, visualized by separating the cleavage products using gel electrophoresis, shows the reactivity of each backbone position toward the radical. This method has been applied to studies of DNA bending and helical twist. Phased runs of adenines (adenine tracts) cause sequence‐directed DNA bending. The hydroxyl radical cleavage of a bent DNA fragment containing short adenine tracts phased with the helix screw gives rise to an unusual cutting pattern. The hydroxyl radical cleavage rate decreases in the 5′ to 3′ direction along each adenine tract, with a minimum at the 3′ end of each adenine tract. The cleavage of the matching thy mine tract is similar, but the minimum in the pattern is offset in the 3′ direction. This pattern on the autoradiograph of the gel is interpreted to indicate that bending is accompanied by a narrow minor groove in the DNA molecule. Furthermore, hydroxyl radical cleavage results in different cutting patterns for two similar sequences, (CGA4T4)5 and (CGT4A4)5, which have been shown to be bent and relatively straight, respectively. The hydroxyl radical method has also been used to determine the helical repeat of the metallothionein IIA gene to be about 10.5 base pairs per turn. Methods of optimizing the hydroxyl radical reaction for DNA‐protein footprinting are discussed. Because individual gel bands give information about cutting frequency at particular positions in the backbone, gel resolution and clear autoradiographs are important to this work. We discuss two problems in these areas which our lab has encountered and solutions we have found for them. Band compressions often occur in G/C‐rich sequences. This can be avoided by adding 25–40 % formamide to 50 % urea denaturing polyacrylamide gels. Drying gels affords clearer autoradiographs of gels, yet higher percentage polyacrylamide gels are difficult to dry or even to remove from the plates. A method has been developed for drying 20–25 % polyacrylamide gels that involves using plastic wrap, instead of filter paper, to remove the gel from the second plate.
Quantitative footprinting analysis of drug‐DNA interactions: Fe (III) methidium‐propyl‐EDTA as a probe
Authors: Dabrowiak James C. , Kissinger Koren , Goodisman Jerry 
Published: May 2005

AbstractQuantitative footprinting studies involving a 139‐base pair restriction fragment from pBR322 DNA, a lexitropsin ligand and two different DNA cleavage agents, the enzyme DNase I and the footprinting reagent Fe(III) methidium‐propyl‐EDTA (Fe‐MPE), are described. The autoradiographic data showed that the ligand, an analogue of netropsin possessing two N‐methylimidazole groups, binds to four regions on the 139‐mer which are rich in GC. Analysis of the data leading to individual binding constants for each of the four loading events on the 139‐mer revealed that Fe‐MPE and DNase I report the same binding constants for the lexitropsin bound to its interaction sequences. The fact that the data from both probes can be analyzed using a common model indicates that the DNA cleavage specificity of the probe and not its binding/ cleavage mechanism is the important factor in reporting of site loading information in the footprinting experiment. The study also showed that under certain conditions it is possible to gain information on the density of ligand binding sites on carrier DNA by monitoring site loading events on the labeled fragment.
Analysis of immune complexes of cerebrospinal fluid by two‐dimensional gel electrophoresis
Authors: Wiederkehr Felix , Wacker Marianne , Vonderschmitt Dieter J. 
Published: May 2005

AbstractFrom the cerebrospinal fluid of 32 patients with different neurological diseases immune complexes were isolated using protein A‐Sepharose. The isolated heavy and light chains and their constituents were analyzed by two‐dimensional gel electrophoresis. In addition to immunoglobulins, some proteins such as albumin, apolipo‐protein A‐I and a number of unknown proteins were detected in all preparations. A complex consisting of three proteins with molecular masses between 52–55 kDa reacted slightly with polyclonal antibodies to glial fibrillary acidic protein, Whether the linkage between these antigens and the Ig is due to the Fab region or the Fc region remains unknown in our study. In some immune complexes of neurological diseases such as amyotrophic lateral sclerosis, astrocytoma and multiple sclerosis, differences are easily recognizable in the gel pattern.
Fixation of amino acids and oligopeptides by freezedrying in polyacrylamide gel electrophoresis
Authors: Nishizawa Hideyuki , Suzuki Keiko , Abe Yoshihiro 
Published: May 2005

AbstractFollowing polyacrylamide gel electrophoresis in an acetic/formic acid buffer, pH 2.0, in fabric reinforced gels, amino acids and oligopeptides could successfully be fixed in the gel by freeze‐drying. Lyophilization of the fabric reinforced polyacrylamide gel after electrophoresis resulted in a dry film which absorbed ninhydrin solution quickly and uniformly, thus improving the detection limit for amino acids and oligopeptides with molecular weights ranging from 189–1045. Most amino acids were detected with a sensitivity of 0.1–0.25 μg and for oligopeptides the detection limit was found to be 0.5–5 μg.