bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic aspects of variation of protein amounts in maize and pea
Authors: de Vienne Dominique , Leonardi Agnès , Damerval Catherine
Published: May 2005

Using high‐resolution two‐dimensional polyacrylamide gel electrophoresis we studied the polymorphism of protein amounts in some genotypes of maize and pea. This type of variability seems to be rather common and insensitive to environmental conditions, as attested by the comparison of the patterns of two maize lines harvested in two different years. A large‐scale experiment involving 5 lines, 7 of their hybrids, and 6 organs (or physiological stages) of maize allowed us to examine numerous polypeptides regrading their genetic variability, their amount differences between organs and the inheritance of their abundance. Genetic and organ variations are not independent: polypeptides whose amount varies from one organ to another are, for the most part, genetically variable (59 %), while the stable polypeptides are not often genetically variable (18 %). We found a striking organ specificity for (i) the extent of quantitative variability (from 2.3–15.4 % of the polypetides), (ii) the occurrence and the type of variation for a given polypeptide (an intensity difference seen in an organ can disappear or even be reversed in another one), (iii) the kind of inheritance (additive/non‐additive): combining the 6 organs and the 7 hybrids we found 101 cases of non‐additivity (4 % of the total) which conecern as many as 72 different spots, that is to say that in most cases a polypeptide displaying nonadditivity in an organ seems to display additivity in the other ones. Moreover, for most of the polypeptides with nonadditive inheritance the hybrid spot presents an intensity similar to that of the most intense parental spot. Thus for protein experssion a hybrid cannot be predicted from its parents. The organ‐specificity was also evidenced by analyzing pleiotropic effect of gene substitutions in various organs of garden pea. Comparisons of isogenic lines differing for the r‐locus revealed that according to the organ, the gene substitution caused appearances, disappearances or quantitative changes for 0 % to more than 10 % of the proteins revealed. All these features are discussed in connection with the possible role of variation of protein amounts in phenotypic variability.
Detection of polypeptides associated with the cold acclimation process in spinach
Authors: Guy Charles L. , Haskell Dale
Published: May 2005

Exposure 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

An 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

Human 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

The 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

We 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

Multiple 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

Optimal 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

An 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

Human 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

Sodium 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

Polyacrylamide 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

A 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

Gels 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

In 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

R‐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

The 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.
Automatic evaluation of nucleic acid sequencing gel autoradiographs
Authors: Ehrhardt Wolfgang , English Uwe , Neuhoff Volker
Published: May 2005

An algorithm for automatic evaluation of nucleic acid sequencing gel antoradiographs is described which is simple and fast, with a low level error rate.
Preparation of plant DNA for separation by pulsed field gel electrophoresis
Authors: Devos Katrien M. , Vercruysse‐Dewitte Daisy
Published: May 2005

A 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

Human 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

The 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

The 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

Quantitative 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

From 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

Following 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.
Heterogeneous nature of human complement factor B: An electrophoretic approach for the analysis of its oligosaccharide chain structure and its physiological breakdown products
Authors: Garnier Gérard , Davrinche Christian , Charlionet Roland
Published: May 2005

Factor B is a glycoprotein which plays an essential role in the alternative pathway of complement activation. It carries the proteolytic activity of the convertases, and its physiological breakdown products Ba and Bb have some effects on the cells of the immune system. Human factor B exhibits a microheterogeneity and five isoforms are present in serum. The nature and origin of the microheterogeneity was investigated by using electrophoretic techniques. Treatments of factor B with neuraminidase and glycopeptidase F show that this microheterogeneity is mainly due to differences in its sialic acid content, varying from seven to eleven residues per molecule, and resulting in different oligosaccharide structures. However, deglycosylated factor B reveals a residual, nonallotypic variation in the Bb region of the polypeptide backbone. We confirm the presence of four asparagine‐linked oligosaccharide chains of the complex type in native factor B, two of which are located in the Ba fragment, and the two others in the Bb fragment. The prevalent isoform of the native protein carries two sialic acid residues per oligosaccharide chain. Biosynthesis experiments show that the microheterogeneity of secreted factor B from HepG2 cells is acquired during the processing of its glycans. However, in vitro‐secreted factor B is more heterogeneous than the serum protein. We propose a structural model for the microheterogeneity of the native protein and its physiological fragments. We discuss as well the feasibility of electrophoretic techniques to deal with microheterogeneity analysis.
Ultrasensitive polychromatic silver staining of sodium dodecyl sulfate‐polyacrylamide gels with the Gelcode system using the PhastSystem Development Unit
Authors: Schiff Theodore A. , Freedberg Irwin M.
Published: May 2005

The Gelcode color‐based silver staining system, an improved formulation based on the original publication by Sammons et al. (Electrophoresis 1981, 2, 141–147) has been adapted to automated rapid staining in the PhastSystem Development Unit. The use of elevated temperatures in the fixation, washing, staining, and stabilization steps of the protocol reduces the total time of the process from 18 h to 1 h. The limit of detection, which is at least tenfold more sensitive than the silver staining protocol recommended for the PhastSystem, corresponds to 0.05–0.1 ng of protein per band. The method is applicable to both one‐ and two‐dimensional polyacrylamide gels.
Specific polypeptide differences in normal versus malignant breast tissue by two‐dimensional electrophoresis
Authors: Wirth Peter J.
Published: May 2005

High resolution two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) in combination with computer‐assisted densitometry was used to analyze 800–1000 silver stained postmitochrondrial and 600–800 cytosolic polypeptides extracted from malignant and nonmalignant human breast tissues. The 2D‐PAGE patterns of polypeptides from malignant and normal tissues were similar, although both qualitative and quantitative polypeptide differences were noted. Six cytosolic poly peptides (pI/ molecular mass X 10−3), 5.20/80, 5.75/43, 6.20/40, 5.43/35, 5.46/34.5, and 5.50/34 were detected exclusively in malignant tissues. One constitutive poly peptide, p52 (7.25/52), was not detected in tumor samples. Marked quantitative differences in spot density were noted in polypeptides localized mainly in the molecular weight ranges of 22–40 kDa and pI of 5.65–7.00. An overall increase in polypeptide expression was noted in this region of 2D‐PAGE gels of malignant tissues as compared to normal. Twenty‐two acidic and 19 polypeptides separated under nonequilibrium isoelectric focusing conditions were significantly increased in tumor samples while one polypeptide was decreased. One polypeptide, p24 (6.15/24), was expressed in greatest concentrations in tumors which also expressed the greatest estrogen receptor content. Expression of p24 was markedly reduced in normal tissue and in malignant tissues expressing low levels of estrogen and progesterone receptors. No significant differences in the expression of the Yb and Ya subunits of glutathione S‐transferases (GST)‐A, ‐B and ligandin were observed between normal and malignant breast tissue. None of the Yp subunits of the placental isoform of GST were detected in either normal or malignant breast tissues.
A procedure for analysis of densitometric spectra
Authors: Galat Andrzej
Published: May 2005

Computer‐aided quantitative analysis of densitometric spectra is presented. The densitometric spectra are decomposed into component bands using the Powell and Marquardt minimizers. Several different functions for the component bands are utilized. It is shown that the densitometric spectra can be decomposed into component bands with high accuracy only if the proper shapes of the bands are chosen. The method described was used for quantitative analysis of densitometric spectra of DNA cleaved by neocarzinostatin. The procedure is general and can be applied to analyses of autoradiographic films and to direct scans of electrophoretic gels. It is shown that densitometric spectra which show highly overlapped bands are well approximated by asymmetric Cauchy and Gauss functions. Well separated densitometric bands which have substantial asymmetry can be fitted to more sophisticated shapes formed by combinations of symmetric and asymmetric Cauchy and Gauss functions. High accuracy fitting to asymmetric densitometric curves may only be achieved using the cosine function introduced by Mignot and Rondot, J. Appl. Cryst. 1976, 9, 460–465.
Distortion of the electrophoretic titration curves of some proteins
Authors: Suttnar Jiří , Dyr Jan Evangelista
Published: May 2005

The electrophoretic titration curves of complex mixtures of vitamin K‐dependent human blood proteins and proteins of Bothrops asper venom were investigated. In both protein mixtures some curves exhibited marked distortions such as additional maxima and minima when Pharmalyte 3–10 carrier ampholytes were used for isoelectric focusing in agarose gels. The distortions result from an unspecific interactions‐between some carrier ampholyte constituents with particular proteins. The interacting carrier ampholyte components could be completely removed by binding to albumin and ultrafiltration through a UM‐2 Amicon membrane with resultant regular titration curves. The interacting carrier ampholyte species were only partially removed by ultrafiltration through a UM‐2 membrane without incubation with albumin.
Isotachophoretic determination of herbicides prometryne, desmetryne, terbutryne and hydroxy‐derivatives of atrazine and simazine in extracts of milk
Authors: Křivánkova Ludmila , Boček Petr , Tekel Jozef , Kovačičová Jana
Published: May 2005

A method is described for the determination of the triazine herbicides prometryne, desmetryne, terbutryne, OH‐atrazine and OH‐simazine in purified extracts of milk using analytical capillary isotachophoresis. The reproducibility of isotachophoretic analyses was 3.5% and the detection sensitivity reached 2 ng. Recovery of triazines from fortified samples of homogenized full milk (0.05 mg/L) was about 65%.
Study of grasshopper meiosis‐associated proteins using two‐dimensional polyacrylamide gel electrophoresis
Authors: Percy Melanie J. , Fletcher Hugh L.
Published: May 2005

Premeiotic and meiotic whole testes from grasshoppers were compared for the presence of meiosis associated proteins using one‐ and two‐dimensional sodium dodecyl sulphate‐polyacrylamide gel electrophoresis. One‐dimensional sodium dodecyl sulphate‐polyacrylamide gels detected differences between premeiotic and meiotic samples but two‐dimensional gels gave more precise results. Isoelectric focusing revealed only one meiosis‐associated protein, while nonequilibrium pH gradient electrophoresis detected five more. It is not known whether these proteins relate to the nuclear aspects of meiosis, or associated cellular changes. These proteins have been electrophoretically purified and monoclonal antibodies are being prepared.
The PhastSystem equipment used for crossed immunoelectrophoresis combined with immunoblotting of coprecipitated monoclonal antibodies as studied with platelet membrane receptor proteins
Authors: Solum Nils Olav , Aakhus Anne‐Margrethe , Pedersen Turid , Gaudernack Gustav
Published: May 2005

The Pharmacia PhastSystem equipment has been used for crossed immunoelectro‐phoresis combined with a technique for immunoblotting with monoclonal antibodies. This miniaturized gel system is compared to the conventional approach using platelet membrane receptor proteins as a model. Whereas in the conventional system the electrophoretic procedure takes place within 20 h, 3 h are adequate for the small gel system. Because of the short second‐dimensional electrophoresis, and only one over‐night incubation, the total electrophoretic and blotting procedure could be reduced from about 48 h to 24 h. The amount of antiserum used during the second‐dimensional electrophoresis could be reduced roughly by a factor of 5. The examples with electrophoresis and immunoblotting using platelet extracts in 1 % Triton X‐100 demonstrate that membrane receptor proteins can be studied even when present as noncovalent complexes. The immunoblotting can be used with monoclonal antibodies that do not function in Western blotting.
Polyacrylamide gel electrophoretic characterization of isoenzymes in the marine algal genus Callithamnion
Authors: Russell Stephen J.
Published: May 2005

Techniques have been developed for the visualisation of a range of enzyme species extracted from field collected marine algal populations, to provide biochemical data in the form of zymogram patterns that closely reflect the genetic identity of these plants. These patterns provide an alternative basis for increasingly accurate recognition to complement that provided by morphology, thus helping to clarify the taxonomic problems which exist in the marine algal genus Callithamnion Lyngbye as currently conceived.
Determination of 4‐methylimidazole in caramel color by capillary isotachophoresis
Authors: Kvasnička František
Published: May 2005

A method for the determination or 4‐methylimidazole in caramel color, based on cationic separation of the sample by capillary isotachophoresis, is described. No pretreatment of the sample is necessary and the detection limit was found to be 5 ppm.
High performance capillary electrophoresis of calmodulin
Authors: Chan Kai‐Foon J. , Chen Wilbur H.
Published: May 2005

The electrophoretic properties of purified calmodulin were investigated. High performance capillary electrophoresis of this Ca2+‐binding protein in free solution at pH 2.5 resulted in an elution of a single peak with a retention time of approximately 4.7 min. Addition of [ethylene‐bis(oxyethylenenitrilo)]N, N, N', N'‐tetraacetic acid (EGTA) to the protein prior to capillary electrophoresis completely abolished this electrophoretic profile. Polyacrylamide gel electrophoresis of calmodulin under denaturing and nondenaturing conditions also revealed a single polypeptide band. However, the relative electrophoretic mobilities of this protein could vary, depending on the presence or absence of Ca2+. The pI of calmodulin was estimated to be 3.7 by using isoelectric focusing techniques. Analysis of this acidic protein by high performance capillary electrophoresis at pH 8.0 revealed that it could be resolved into two major and one minor polypeptide peaks, regardless of the presence or absence of Ca2+. These findings suggest that capillary electrophoresis at near physiological pH may differentiate the microheterogeneity of calmodulin.
Electrophoretic separation of protein kinases: Altered mobility with different crosslinking agents in the presence of certain detergents
Authors: Sprott Susan , Hammond Kathryn D. , Savage Nerina
Published: May 2005

Nondenaturing polyacrylamide gel electrophoresis was used to separate protein kinases in crude extracts and subcellular fractions of murine erythroleukemic cells. The kinases were detected using an in situ phosphorylation assay. The electrophoretic patterns obtained using gel bound to GelBond and prepared with AcrylAide differed from those seen without GelBond and with N, N'‐methylenebisacrylamide as cross‐linker. In an attempt to improve the resolution of the bands in the membrane fractions, detergent‐treated preparations were electrophoresed through gels which contained either 0.1 % Triton X‐100 or 0.1 % Nonidet P‐40. The resolution of the bands in this fraction was not, however, improved with the inclusion of the nonionic detergent in the gels. When cytosol was electrophoresed through gels containing detergent, a major band of cAMP‐dependent protein kinase activity showed a marked shift in mobility. This may have been the result of a structural change, altering the shape and possibly affecting the charge on the molecule, or the enzyme may have formed aggregates with the detergent.
Reassessment of commercially available molecular weight standards for peptide sodium dodecyl sulfatepolyacrylamide gel electrophoresis using electroblotting and microsequencing
Authors: Sallantin Marc , Huet Jean‐Claude , Demarteau Claude , Pernollet Jean‐Claude
Published: May 2005

Myoglobin CNBr peptides, constituting the commercially available molecular weight calibration kits for sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, were analyzed by microsequencing after electroblotting on polyvinylidene difluoride (Immobilon) membranes. An obious disagreement was found between peptide identification and the data provided by the manufacturers. We observed 6 peptides from Mr 2500 to 17 000 corresponding, in increasing size order, to the 3 peptides resulting from the total CNBr digestion, to 2 incompletely cleaved peptides and to the intact myoglobin. Using a corrected calibration curve, a linear relationship was established from Mr 6000 to 43 000 and a second one for shorter peptides. This method of electrophoresis and electroblotting, easily adapted for peptides, is a powerful tool for peptide identification correlated with size determination. It is especially useful for CNBr‐cleaved peptides.
Microheterogeneity of human erythrocyte pyruvate kinase: Application of immunological visualization techniques after isoelectric focusing in ultrathin gels
Authors: Megow Dirk , Jacobasch Gisela
Published: May 2005

Immunological methods for protein visualization afford high sensitivity, good specificity and resolution. We used the indirect immunoassay with peroxidase‐ and 125I‐labelled antibodies to investigate the microheterogeneity of pyruvate kinase from human red blood cells and can show an enzyme pattern with more than 10 single bands. Different methods were tested as to their applicability for protein fixation and subsequent immunological visualization in polyacrylamide gels. Besides the Western blot, immunofixation and ethanolic fixation can be recommended as fixation techniques, especially after isoelectric focusing in ultrathin gels.
Molecular characterization of pregnancy‐associated plasma protein‐A by electrophoresis
Authors: Sinosich Michael J.
Published: May 2005

Gradient polyacrylamide gel electrophoresis, isoelectric focusing and multidimensional immunoelectrophoretic techniques have been applied in order to physicochemically characterize pregnancy‐associated plasma protein‐A (PAPP‐A). By lectin affinity immunoelectrophoresis, PAPP‐A contained sialic acid, glucose/mannose and N‐acetyl‐α‐D‐galactosamine. Immunoelectrophoretic analyses after incubation with various glycolases confirmed these findings and demonstrated that PAPP‐A contained glucuronic acid, perhaps in chondroitin sulphate moities, thus indicating that PAPP‐A may be a proteoglycan rather than a glycoprotein. Analysis by metal chelate and dye ligand affinity immunoelectrophoresis demonstrated many similarities between PAPP‐A and α2‐macroglobulin (α2M). However, unlike α2M, PAPP‐A did not form immunologically reactive complexes when incubated with proteases. Furthermore, as demonstrated by autoradiographic studies, PAPP‐A did not contain internal thiolester groups, thus indicating that PAPP‐A cannot inhibit proteases by molecular entrapment and, despite the homotetrameric molecular conformation, PAPP‐A and α2M may not have evolved from a common ancestral protein.
Horizontal two‐dimensional electrophoresis of cerebrospinal fluid proteins with immobilized pH gradients in the first dimension
Authors: Mattila Kari , Pirttilä Tuula , Frey Harry
Published: May 2005

A horizontal two‐dimensional electrophoresis method with immobilized pH gradient isoelectric focusing supplemented with carrier ampholytes in the first dimension was applied to cerebrospinal fluid (CSF) proteins. About 300 protein spots could be detected on the silver‐stained two‐dimensional maps of CSF samples. This highresolution method is a tool worthy of consideration for the research of CSF proteins and disease‐specific changes in different neurological disorders.
Alternative methods for fixing and staining gliadins in polyacrylamide gels
Authors: Clements Robert L.
Published: May 2005

Staining efficiencies of Coomassie Brilliant BlueG‐250 (CBB G‐250) and Coomassie Brilliant Blue R‐250 (CBB R‐250) in various media were studied in efforts to reduce or eliminate requirements for trichloroacetic acid (TCA). Stained gels were compared with gels stained with CBB R‐250 in 12% TCA and evaluated for overall stain and background. Because of qualitative effects, stain intensities of low‐ and highmobility gliadins were also evaluated. Results indicated gliadins are fixed under a wide range of conditions, permitting adjustment of conditions to provide optimum staining. CBB G‐250 and R‐250 in tap water fixed and stained most gliadins. Best results were obtained with CBB G‐250 in 2% TCA, in 2% TCA containing 5% sodium sulfate, and in 2% and 5% phosphoric acid containing 5% sodium chloride or 5% sodium sulfate. Gels stained in these media were more easily observed during staining and more easily destained than gels stained in CBB R‐250 in 12% TCA.
Column isoelectric focusing in natural pH gradients generated by biological buffers
Authors: Vaidya Narendra R. , Gothoskar Balwant P. , Banerji Ashutosh P.
Published: May 2005

Using 2 or 3 simple Good z witterionic buffers at a 16 or 18 mmol/L final column concentration of the mixture, natural pH gradients of 4 to 8 and 3 to 9.5, respecitively, were generated in a liquid LKB column. The pH gradients, stabilized by an anticonvective sucrose gradient, were linear, reproducible and stable in the electric field up to 5h. The pH gradients were used for isoelectric focusing of a number of impure proteins such as human hemoglobin, bovine serum albumin and chicken egg white lysozyme. The protein components could be well separated in the gradient, were easily recovered and appeared to be quite pure when analyzed by sodium dodecyl sulfategel electrophoresis. Furthermore, the pH gradient 4–8 was effectively used to isolate one of the acidic isozyme (pI 5.6) components of mouse liver alcohol dehydrogenase (EC 1.1.1.1) in an enzymatically active state, suggesting that the procedure does not denature proteins. The low cost, the ease with which the pH gradients are formed, their linearity, stability for a sufficient period to allow proteins to reach equilibrium and their subsequent recovery from buffer eluates should make the procedure interesting for electrofocusing of proteins.
Isotachophoretic zone formation of serum albumin in different free fluid electrophoresis instruments
Authors: Thormann Wolfgang , Firestone Millicent A. , Sloan Jeffrey E. , Long Terry D. , Mosher Richard A.
Published: May 2005

The isotachophoretic behavior of a model protein, serum albumin, was examined (i) by computer simulation, (ii) by capillary isotachophoresis in HPE 100 and Tachophor 2127, (iii) by continuous flow isotachophoresis in Elphor VaP 22 and the BIO‐STREAM Separator and (iv) by recycling isotachophoresis in an apparatus of our own design. Variations in monitored zone shapes can be explained by differences in engineering aspects and fluid stabilization principles of the instruments.
Phenotyping of erythrocyte acid phosphatase and esterase D by high field strength isoelectric focusing on cellulose acetate membrane
Authors: Kane Masateru , Yamamoto Yoshio , Yamada Mitsuko , Fukunaga Tatsushige , Tatsuno Yoahitsugu
Published: May 2005

Phenotyping of erythrocyte acid phosphatase (EAP) and esterase D (ESD) by cellulose acetate membrane isoelectric focusing (CAM‐IEF) under a nonequilibrium condition is described. In an attempt to improve the method of CAM‐IEF, we shortened the electrode distance to provide a higher field strength at a given (low) voltage. Various carrier ampholytes for EAP typing and various chemical separators for ESD typing were also tested. Good separations were obtained after 30 min IEF for EAP typing and 25 min for ESD typing. When applied to blood stains and stored for various periods at room temperature, the stains up to 8 months old could still be phenotyped for EAP and those up to 4 weeks old for ESD. CAM‐IEF is suitable for routine forensic work of EAP and ESD phenotyping.