bluuebunny/crossref_metadata_2025 huggingface dataset:
Application of immobilized pH gradient isoelectric focusing to forensic hemogenetics: A survey on a three year experience with the transferrin (TF) and alpha 1‐antitrypsin (PI) systems
Authors: Skoda Ulrike , Faßbender Lydia , Händler Christa , Mauff Gottfried , Pulverer Gerhard
Published: May 2005

The subtypes of transferrin (TF) and alpha 1‐antitrypsin (PI), first discovered using isoelectric focusing, are now mostly determined in immobilized pH gradient gels. We report on our experience in the parentage expertise with both polymorphisms over a period of three years. The complexity of the technology was compensated by the fact that most subtypes of TF and PI could be more reliably recognized. The PI alleles PI*M1, M2, M3, S, F, T, and Z and TF alleles TF*C1, C2 and C3, and in addition four further rare TF alleles were observed. The allele frequencies from non‐related individuals did not deviate from the Hardy‐Weinberg equilibria and corresponded well to known frequencies from West Germany and other Caucasoid populations. With the TF system 36 accused men, and with the PI system 54 were excluded from paternity from a total of 344 (TF) respectively 347 (PI) cases. From the data presented here isoelectric focusing in immobilized pH gradient gels appears to be a major improvement over carrier ampholyte generated pH gradients in the distinction of TF and PI phenotypes.
Isoenzyme patterns as a tool in taxonomy of chlorococcal algae
Authors: Kilias Harald
Published: May 2005

From isolated lichen algae of the genusTrebouxia, De Puymaly isoenzymes of phosphoglucomutase, malate dehydrogenase and superoxide dismutase were studied by isoelectric focusing in hybrid immobilized pH gradients. Phosphoglucomutase has proven to be a suitable marker since it is highly variable whereas malate dehydrogenase and superoxide dismutase are less variable. The focusing patterns are species specific and provide a basis for a reevaluation of species classification ofTrebouxia.
Genetic polymorphisim of a lymphocyte protein (p75) with six different alleles studied by two‐dimensional electrophoresis: Qualitative and quantitative data
Authors: Waldinger Dorothea , Cleve Hartwig
Published: May 2005

The genetic polymorphism of a human lymphocyte protein (p75) was studied by two‐dimensional electrophoresis within 17 families and, in addition, 22 unrelated individuals from Southern Germany, resulting in a total of approximately 100 individuals, The cytosolic and membrane proteins from cell lysates of phytohemagglutinin stimulated and [3H] leucine labeled lymphocytes were separated by two‐dimensional electrophoresis. The p75 protein with an approximate molecular weight of 75 000 occurred in six variants with slightly different isoelectric points and/or apparent molecular weights. Three common variants (a, b, and c) and three rare variants (d, e and f) could be distinguished. Among the approximately 100 individuals studied we observed 15 different phenotypes, three homozygous (p75‐a, ‐b, ‐c) and 12 heterozygous (p75‐ab, ‐ac, ‐bc, ‐ad, ‐ae, ‐be, ‐bf, ‐cd, ‐ce, ‐cf, ‐de, ‐df) phenotypes. The genetics of the p75 protein variations was ascertained by family studies and quantitative computer analysis. We were able to show a Mendelian mode of inheritance of the variants within the families and a gene‐dosage dependence of the protein spots in homozygous and heterozygous phenotypes. The data allowed us to assume a polymorphic protein p75 determined by six alleles on a autosomal gene locus. The allele frequencies were calculated from the phenotype distribution within 56 unrelated individuals. The gene frequencies of the three common alleles ranged between 0.38 and 0.22 and the gene frequencies of the three rare alleles ranged between 0.01 and 0.07.
The use of ultrathin‐layer polyacrylamide gel isoelectric focusing in two‐dimensional analysis of plant and fungal proteins
Authors: Marlow Gary C. , Wurst Denise E. , Loschke David C.
Published: May 2005

Although use of ultrathin polyacrylamide gel isoelectric focusing in the first dimension of two‐dimensional analysis bestows a number of advantages, it has been little used by the plant science community. Nonstandardization along with problems unique to the format have probably delayed wider adoption. Relevant parameters were therefore tested in order to optimize resolution, reproducibility, economy and ease of use. Ultrathin‐layer gels (200 μm in this study) used in the first dimension require a semirigid backing for support. Widely available matte‐finished thin polyester film without chemical pretreatment was found to bind the gel adequately. The gel adheres to the film through all processing steps, yet, if desired, can be easily transferred to Whatman 3MM paper for special applications such as Western blotting. The ultrathin first‐dimensional gels can be quickly dried on the polyester backing for convenient handling and long term storage. Strips cut from the dried gel for use in the second dimension are more easily manipulated than their tube format counterparts. The difficulty of disrupting and recovering microsamples of labeled leaf and root tissue prompted the invention of an efficient and simple communition device. An economical and efficient silver stain process is also described. This analytical technique was applied in an attempt to detect resistance gene products in different genetic backgrounds of maize. Although the ultrathin flatbed format provides as good as or better resolution than the tube gel system, the level of sensitivity was still inadequate to reveal the apparently rare resistance gene product.
Significant immunological cross‐reactivity of plant glycoproteins
Authors: Laine Anne‐Catherine , Faye Loïc
Published: May 2005

Plant glycoproteins generally cross‐react because of the presence of identical or related complex glycans which are highly immunogenic. The use of mild periodate oxidation of glycans after glycoprotein transfer from sodium dodecyl sulfate‐poly‐acrylamide gel electrophoresis gels to nitrocellulose membranes prior to immunodetection is a way of identifying the carbohydrate antigenic determinants of a glycoprotein as the basis for antigenic cross‐reaction. Periodate oxidation can distinguish between antibodies directed against carbohydrate and against peptide antigenic determinants, the latter being unaffected by oxidation. Immunoblotting performed after periodate treatment allows the detection of common protein epitopes.
Multi‐chamber apparatus for preparative isoelectric focusing
Authors: Kyhse‐Andersen Jan
Published: May 2005

A multi‐chamber apparatus for preparative isoelectric focusing is described. The apparatus is constructed of 32 separation chambers and 2 electrode chambers, all separated by uncharged porous membranes. The total volume of the 32 separation chambers is 660 mL. A cooling system and a stirring system are built in. Human serum proteins were separated by isoelectric focusing in a natural pH gradient. The fractionation was monitored by fused rocket immunoelectrophoresis. The number of fractionation was monitored by fused rocket immunoelectrophoresis. The number of proteins in each fraction was monitored by crossed immunoelectrophoresis. The apparent pI values of IgG. transferrin and alpha‐1‐antitrypsin are as found in the literature. Orosomucoid (alpha‐1‐acid glycoprotein) (pI = 1.8) is concentrated at the acid end of the pH gradient.
Identification of different classes of superoxide dismutase in carnation petals
Authors: Droillard Marie‐Jo , Bureau Danielle , Paulin André , Daussant Jean
Published: May 2005

Isoelectric focusing and nondenaturing two‐dimensional electrophoresis, followed by an enzymatic characterization involving use of specific inhibitors were applied to identify the different forms of the superoxide dismutase (SOD) extracted from petals of cut carnation. Electrophoresis was carried out either with the crude extract or with fractions obtained after ion exchange chromatography of the extract. Three forms of the SOD were identified (Cu, Zn‐SOD, Fe‐SOD and Mn‐SOD). They have close pI's (4.7 to 4.9) and most of them are composed of several isoforms.
Database and search techniques for two‐dimensional gel protein data: A comparison of paradigms for exploratory data analysis and prospects for biological modeling
Authors: Lemkin Peter F. , Lester Eric P.
Published: May 2005

Two‐dimensional (2‐D) polyacrylamide gel electrophoresis can detect thousands of polypeptides, separating them by apparent molecular weight (Mr) and isoelectric point (pI). Thus it provides a more realistic and global view of cellular genetic expression than any other technique. This technique has been useful for finding sets of key proteins of biological significance. However, a typical experiment with more than a few gels often results in an unwieldy data management problem. In this paper, the GELLAB‐II system is discussed with respect to how data reduction and exploratory data analysis can be aided by computer data management and statistical search techniques. By encoding the gel patterns in a “three‐dimensional” (3‐D) database, an exploratory data analysis can be carried out in an environment that might be called a “spread sheet for 2‐D gel protein data”. From such databases, complex parametric network models of protein expression during events such as differentiation might be constructed. For this, 2‐D gel databases must be able to include data from other domains external to the gel itself. Because of the increasing complexity of such databases, new tools are required to help manage this complexity. Two such tools, object‐oriented databases and expert‐system rule‐based analysis, are discussed in this context. Comparisons are made between GELLAB and other 2‐D gel databases analysis systems to illustrate some of the analysis paradigms common to these systems and where this technology may be heading.
Development of a database of amino acid sequences for proteins identified and isolated on two‐dimensional polyacrylamide gels
Authors: Sweatt J. David , Kennedy Timothy E. , Wager‐Smith Karen , Gawinowicz Mary A. , Barzilai Ari , Karl Kevin A. , Kandel Eric R.
Published: May 2005

As part of our continuing studies into the biochemical basis of long‐term changes in neuronal function in Aplysia, we have developed a simple method for obtaining amino acid sequence information from proteins isolated on two‐dimensional gels. Proteins isolated on preparative two‐dimensional gels are digested in situ with Staphylococcus, aureus V8 protease, and the resulting peptides electrophoresed, transferred to a polyvinylidene difluoride membrane, and sequenced in a gas‐phase sequencer. The method is simple and should be applicable to a variety of other systems where the development of a two‐dimensional gel database is underway.
A simple method for determination of the concentration of anti‐dextran IgG in antiserum by means of affinity electrophoresis
Authors: Tanaka Tatehiko , Nakamura Kazuyuki , Takeo Kazusuke
Published: May 2005

A simple technique for determination of the concentration of anti‐dextran IgG in antiserum using affinity electrophoresis is described. In the presence of an excess of intermediary molecular size dextran in the polyacrylamide gel, polyclonal anti‐dextran IgG migrated in a single sharp band, separated from the nonspecific IgG fraction and other serum protein fractions. With this technique, 1–10 μg anti‐dextran IgG in antisera can be determined within 3 h. We call the procedure ligand saturating affinity electrophoresis.
Characterization of human residual catalase of an acatalasemic patient by isoelectric focusing and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis followed by electrophoretic blotting and immunodetection
Authors: Ogata Masana , Suzuki Kazuhiko , Satoh Yukinori
Published: May 2005

Isoelectric points and subunit sizes of catalases in human blood and human cultured skin fibroblasts from acatalasemic and normal subjects were analyzed by isoelectric focusing in agarose gel and by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, respectively, followed by electroblotting to polyvinylidene difluoride membranes for immunodetection. The results indicated that the isoelectric point of residual catalase in the C fraction prepared from acatalasemic erythrocytes was identical with that of catalase prepared from normal erythrocytes. The residual catalase in homogenates of acatalasemic cultured skin fibroblasts also reacted with anticatalase rabbit serum and had an isoelectric point identical with that of normal catalase. Sub‐unit sizes of normal and acatalasemic catalases in the C fractions of erythrocytes were also found to be identical on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, followed by electroblotting and immunoenzymatic amplification. The results indicated no substantial difference in molecular size and charge of catalase proteins between normal and acatalasemic erythrocytes and fibroblasts.
Use of a polynomial exponential function to describe migration of proteins on sodium dodecyl sulfate‐polyacrylamide gels
Authors: Winston Vern
Published: May 2005

Standard mixtures of proteins were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Polynomial regression analysis was used to fit curves to the data, points obtained by plotting log10 of protein molecular weight versus electrophoretic mobility. Polynomials with orders ranging from 1 to 4 were generated. The coefficients of each equation were analyzed for statistical significance. It was found that a third order polynomial was the hightest‐order equation in which all coefficients contributed significantly to the prediction of molecular weights. Using this equation, it was possible to estimate the molecular weights of known proteins in the range from 97 400 to 14 400 with a maximum error of 1 %, compared with a maximum error of 17 % when a first‐order equation was used to describe the migration of the standarads.
Interference of the carbohydrate moiety in Coomassie Brilliant Blue R‐250 protein staining
Authors: Osset Miquel , Piñol Montserrat , Fallon Martin J. M. , De Llorens Rafael , Cuchillo Claudi M.
Published: May 2005

The binding of Coomassie Brilliant Blue R‐250 to several species of bovine pancreatic ribonuclease is affected by the presence of a carbohydrate moiety in the enzyme molecule. Enzymic deglycosylation of several chromatographic fractions of ribonuclease, which have different degrees of glycosylation, results in increased staining by Coomassie Brilliant Blue R‐250. Ovalbumin and other glycoproteins tested show similar behavior. The results indicate that carbohydrate moieties may represent a common hindrance to the binding of Coomassie Brilliant Blue dyes to glycoproteins.
Pulsed field gel electrophoresis: Studies of DNA migration made with the programmable, autonomously‐controlled electrode electrophoresis system
Authors: Birren Bruce W. , Hood Leroy , Lai Eric
Published: May 2005

We have studied the migration of DNA in pulsed field agarose gels under a variety of electrophoresis conditions. We have made use of an instrument which can generate electric fields of any orientation, magnitude, or duration to compare different separation techniques for DNA molecules of from 1 to several thousand kilobase pairs. We discuss the capabilities of the system and present results of gel runs in which electrophoresis conditions were changed individually or in combination. The mobility of DNA in pulsed field gels is shown to reflect a number of interdependent physical parameters.
Separation of chromosomal length DNA molecules: Pneumatic apparatus for rotating gels during electrophoresis
Authors: Sutherland John C. , Emrick Ann B. , Trunk John
Published: May 2005

The maximum length of DNA molecules that can be separated by gel electrophoresis can be increased greatly by periodically altering the direction of the electric field with respect to the gel by an angle that exceeds 90°. One method involves rotating the gel by the desired angle in alternate directions periodically during electrophoresis. We describe a modification of the rotating gel electrophoresis apparatus developed by Serwer (Electrophoresis 1987, 8, 301–304) that uses a pneumatic rotary actuator instead of a stepping motor, hence reducing the cost by about 50 %. Other advantages of our design are a lower center of gravity that makes the apparatus more stable and the removal of all electrical power from beneath the fluid‐filled electrophoresis chamber. We present data demonstrating the separation of chromosomal length DNA molecules from Saccharomyces cerevisiae strain 334 into 14 resolved bands in parallel lanes.
Gel electrophoretic analysis of DNA branched junctions
Authors: Seeman Nadrian C. , Chen Jung‐Huei , Kallenbach Neville R.
Published: May 2005

Gel electrophoresis has provided much of the detailed information we have about the properties of DNA junctions, stable branched molecules formed from oligonucleotide or polynucleotide strands. Here we review these applications, and present the results of an electrophoretic investigation of conformationally restricted junctions formed by covalently connecting two different pairs of strands in a junction with four arms. Native gel electrophoresis is employed to establish the formation and stoichiometry of the multistrand complexes. Ferguson analysis of native gel mobility shows that junctions have retardation coefficients that are distinct from those of linear DNA duplexes.

Denaturing gel electrophoresis is the primary tool for characterizing junctions that have been covalently linked together to form both linear and macrocyclic oligomers of junctions (oligojunctions). Radioactively labelled strands enable one to monitor the progress of the ligation reaction: both linear and closed cyclic molecules result, and these can be distinguished by applying Ferguson analysis to denaturing gels. Combinations of exonuclease III, restriction enzymes and sequencing reactions have been applied to oligojunction molecules, and the results are all analyzed on denaturing gels.

Junctions containing intramolecular “tethers” that restrict the conformation freedom of the complex comprise a new system for analyzing the conformations of branched molecules. In these tethered junctions, the ability of arms to moverelative to each other is restricted substantially by covalently connecting pairs of arms in the original complex with short, flexible loops. The two tethers used here constrain the helical domains of the structure to be roughly parallel or anti‐parallel. In this article we use Ferguson analysis to compare two tethered junctions with an untethered junction. At high gel concentrations, the mobility of the untethered complex is found to be closer to that of the molecule tethered anti‐parallel than to the one tethered parallel. Curvature in the Ferguson plots for all three of these junctions is detected over arange of compositions. At low gel concentrations, differences in electrophoretic mobility persist, suggesting that the untethered junction differs in charge as well as conformational freedom from the tethered analogs. We expect that studies of this kind will able to define the conformational repertoire of junctions of different kinds, and to explore the effects of electrophoresis on these states.
Studies of DNA‐protein interactions by gel electrophoresis
Authors: Ceglarek John A. , Revzin Arnold
Published: May 2005

The use of gel electrophoresis in studies of nucleic acid‐protein (especially DNA‐protein) interactions has yielded much qualitative and quantitative information about a variety of such systems. The reduction in mobility of complexes relative to free DNA allows isolation and characterization of the complexes as well as determination of thermodynamic and kinetic properties of the interactions. This article begins with a review of recent applications of the “gel retardation” assay, by way of introduction to experiments in two areas. In the first, a hypothesis is tested regarding whether a DNA molecule with sizable proteins bound very near to each end migrates through a polyacrylamide gel differently than does the corresponding complex having the proteins in the middle of the DNA fragment. The data show little mobility differences for these types of complexes, implying that both may move in a linear, “snakelike”, manner through the gel. The experiments also provide results pertaining to questions of DNA bending caused by the binding of the E. coli catabolite activator protein (CAP) and RNA polymerase to the lactose promoter region. It appears that DNA bending by CAP at its wild type lac binding site is retained in complexes where RNA polymerase is bound simultaneously at the lac UV5 promoter.
The use of denaturing gradient gel electrophoresis to screen for DNA sequence polymorphisms in the human factor VIII gene
Authors: Collins Mary , Wolf Stanley F. , Haines Lora L. , Mitsock Lisa
Published: May 2005

We describe the use of denaturing gradient gel electrophoresis to screen for DNA sequence polymorphisms in the human factor VIII gene. DNA fragments that differ in sequence by only a single base pair can be separated on denaturing gradient gels due to changes in their melting behavior. Previous studies have demonstrated the use of denaturing gradient gels to detect sequence changes in human genomic DNA, including mutations in the β globin gene and polymorphisms on chromosome 20. We have begun to use denaturing gradient gels to look for polymorphisms within the human factor VIII gene. The DNA sequences of seven cloned fragments from introns in the human factor VIII gene were determined and used to predict a melting map for each fragment. The melting behavior of each cloned fragment was evaluated by electrophoresis into denaturing gradient gels. Appropriate fragments were then used as radioactive probes for hybridization to human DNA samples that had been digested with restriction enzymes. Heteroduplexes formed between the probe and genomic DNA samples were electrophoresed into denaturing gradient gels. The final positions of heteroduplex bands were determined by autoradiography. We describe a general approach for using denaturing gradient gel electrophoresis to find DNA polymorphisms, with particular emphasis on the predictive value of DNA sequence data. We compare the efficiency of polymorphism detection by denaturing gradient gel electrophoresis with detection by restriction fragment length polymorphism (RFLP) analysis. The factor VIII gene appears to have a low level of DNA sequence polymorphism. We detected only two rare sequence variants using seven different probes to screen almost 2500 base pairs from each of at least 12 different chromosomes. This low level of sequence polymorphism is consistent with other measurements suggesting that nucleotide variations occur less frequently on the X chromosome than on autosomes.
Reptation‐Breathing theory of pulsed electrophoresis: Dynamic regimes, antiresonance and symmetry breakdown effects
Authors: Viovy Jean L.
Published: May 2005

We apply the concepts of tube and reptation to the pulsed electrophoresis of DNA, considering both biased reptation and “breathing” modes (internal modes of the chain). Using suitable preaveraging approximations, analytical expressions are derived which relate displacement in crossed field electrophoresis to molecular weight, field strength, field period, pore size of the gel, and the angle between the field. These expressions provide scaling laws for the change of mobility when one (or more) of the parameters is varied as well as “universal” velocity versus molecular weight versus pulse time curves. These results are quantitatively compared with experiments. At some point which depends on field angle, field strength and chain length, however, we predict a failure of this model due to symmetry breakdown and loss of ergodicity. Qualitatively, this should lead to considerable band spreading and/or splitting of the highest DNA bands into two bands migrating sideways from the diagonal. The case of field inversion is also investigated. It is shown that only breathing modes can explain the strong differences in mobility experienced by chains of different length when opposite fields of equal amplitude are applied: the “trapping” of chains in conformations of low mobility is associated with an antiresonance‐like coupling between the external field and the internal modes.
Elevated plasma levels of haptoglobin in duchenne muscular dystrophy: Electrophoretic variants in patients with a severe form of the disease
Authors: John Huw A. , Purdom Ian F.
Published: May 2005

Analysis of plasma proteins of Duchenne, muscular dystrophy (DMD) patients and normal age‐matched boys using two dimensional gel electrophoresis and densitometry indicated a three‐fold increase in the average haptoglobin level in DMD plasma. Two electrophoretic variants of haptoglobin in which the α2 chain had more basic or more acidic spots were found in DMD patients with a rapidly progressing form of the disease. The basic variant was present in relatively low amount. Possible reasons for the elevated haptoglobin levels are discussed.
Transparency of nitrocellulose membranes with triton X–114
Authors: Vachereau Andre
Published: May 2005

Transparency of nitrocellulose membranes can be realized by simply impregnating the filters with concentrated Triton X‐114. Within a few seconds, protein blots become transparent and can be photographed by transillumination or scanned on a densitometer. Removal of the detergent is possible and the recovered filters can further be used for immunodetection purposes. This procedure is particularly well‐suited where extensive investigation of a single separated protein sample is required.
A two‐dimensional electrophoresis procedure for single meristems of different forest species
Authors: Bon Marie‐Claude
Published: May 2005

High resolution two‐dimensional electrophoresis, with isoelectric focusing in the first dimension and electrophoresis in sodium dodecyl sulfate in thin acrylamide gels in the second dimension, has been applied to separate the proteins of single meristems (200‐300 μm) from Sequoiadendrongiganteum, Sequoiasempervirens, Pseudotsuga menziesii, Picea abies, Pinus pinaster, Eucalyptus gunnii and Populus nigra. The technique may prove an efficient tool for studying inter‐ and intraclonal differences in meristem cultures of forest species, especially when referring to cloning from meristems of selected trees in relation to the rejuvenation process.
Two‐dimensional electrophoretic analysis of nuclear proteins from human tumors
Authors: Menzel Alain , Unteregger Gerhard
Published: May 2005

During the last decade several strategies have been developed to identify proteins which could serve as markers in tumor biology. One avenue of great promise to detect such proteins seems to be the separation of prefractionated organelles from tumor cells by high resolution two‐dimensional electrophoresis. Using detergent‐lysed nuclei from several human tumor cell lines, especially from brain tumors, and two‐dimensional electrophoresis, we analyzed the nuclear protein pattern obtained after sequential salt extraction of tumor cell nuclei. In addition to proteins occurring in all tumor cell lines, the pattern of different tumor cell lines exhibits considerable differences when proteins were visualized by silver staining, thus emphasizing the specificity of nuclear proteins with respect to the cell type. Even quantitative variations of the nuclear phosphoproteins 23/4 were detectable, indicating a potential correlation between their synthesis/phosphorylation and the proliferation behavior of tumor cells. The data indicate that nuclear proteins with their distinct heterogeneity and tissue specificity may represent a powerful source in determining tumor‐specific proteins. The extent of chromosomal protein heterogeneity may be additionally increased by their covalent modification by nuclear kinases; therefore, tumor‐specific nuclear proteins may occur as quantitative and qualitative variations.
Lectin affinity electrophoresis of α‐fetoprotein in cancer diagnosis
Authors: Taketa Kazuhisa , Hirai Hidematsu
Published: May 2005

Lectin affinity electrophoresis of serum α‐fetoprotein (AFP) was carried out on samples obtained from patients with benign and malignant diseases and on cord blood, and separated AFP bands were detected by antibody‐affinity blotting. The following major bands were identified by determination of kinetic constants: AFP‐C1 and ‐C2 with concanavalin A, AFP‐LI, ‐L2 and ‐L3 with Lens culinaris agglutinin A, AFP‐P1, ‐P2, ‐P3, ‐P4 and ‐P5 with erythroagglutinating phytohemagglutinin, and AFP‐A1, ‐A2 and ‐A3 with Allomyrina dichotoma lectin. AFP bands with the lowest number had either low or no affinity and those with higher numbers had higher affinities for respective lectins. AFP from cord blood and chronic liver disease was characterized by the predominance of AFP‐C2, AFP‐L1, AFP‐P2 and AFP‐A3. Hepatocellular carcinoma was differentiated from the benign liver disease by increased proportions of AFP‐L3 and AFP‐P4. Extrahepatic tumors had additional increases of AFP‐C1, AFP‐L2, AFP‐P5 and AFP‐A1 (or slow‐migrating AFP‐Als, particularly in yolk sac tumor).
Electrophoretically silent hemoglobin mutants as revealed by isoelectric focusing in immobilized pH gradients
Authors: Righetti Pier Giorgio , Gianazza Elisabetta , Bianchi‐Bosisio Adriana , Wajcman Henri , Cossu Gianfranco
Published: May 2005

The applications of isoelectric focusing in immobilized pH gradients to the analysis of (i) human hemoglobin mutants, (ii) animal hemoglobin mutants (from cattle, sheep, dog and mouse), and (iii) tryptic digests of α and β chains, are discussed and evaluated. Immobilized pH gradients appear to be an excellent tool for screening of genetic polymorphism and for detecting “silent mutants”,i. e. those substitutions involving amino acids with nonionizable side chains. At present, not even capillary zone electrophoresis, claimed to have a resolving power equivalent to 1 million theoretical plates, has shown a resolution capability comparable to that of immobilized pH gradients, at least in the field of protein separation.
Separation of different forms of transferrin by isoelectric focusing to detect effects on the liver caused by xenobiotics
Authors: Petrén Sven , Vesterberg Olof
Published: May 2005

Several genetic variants and also isoforms of transferrin differing in carbohydrate structure can be separated by polyacrylamide or agarose gel isoelectric focusing. Numerous blood plasma or serum samples can be analyzed in parallel in each gel. Studies of the heterogeneity of transferrin have already revealed many results of importance to different fields of human medicine. Gene typing can give important and useful information for paternity determination and in forensic medicine. The gene type C 2 seems to have increased frequency in certain malfunctions. Furthermore, functional abnormalities of liver cells can be revealed by determination of the concentrations of transferrin isoforms differing mainly in their carbohydrate parts. The isoforms can be quantified with zone immunoelectrophoresis assay. Thus valuable information can be obtained about important modulated regulations of cell and membrane functions, even when these are disturbed by disease and xenobiotics. The information may be usefule.g. in the detection of individuals suffering from toxic effects, to identify toxic agents and exposure conditions. Studies of house painters revealed that exposure to different types of paints had an effect on transferrin. Determination of the concentration of the isotransferrin with pI5.7 in blood samples from alcoholics can be used as a marker for the detection of liver dysfunction and for the monitoring of therapy treatments. In addition, by analyzing the isotransferrins a rare genetic abnormality can be detected.
Electrophoretic studies of antimitochondrial antibodies: Identification of mitochondrial antigens specific to primary biliary cirrhosis
Authors: Ishii Hideo , Muno Daisaku , Namihisa Toshihiko
Published: May 2005

Mitochondrial inner membrane proteins extracted from beef heart tissue were examined for reactivity to antimitochondrial antibody (AMA) present in sera of patients with primary biliary cirrhosis (PBC) by an immunoblotting technique. Four proteins, which reacted with AMA, had molecular masses of 70 kDa, 50 kDa, 47 kDa and 40 kDa, as defined by their relative mobility (Rf) in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. All sera of 114 PBC patients were positive with at least one and as many as four of the mitochondrial proteins. The major antigenic proteins of mitochondrial inner membrane to which AMA reacts were the 70 kDa and 47 kDa proteins. All PBC sera containing antibodies to the 50 kDa and/or 40 kDa proteins reacted with 70 kDa as well. The isolation of antigen reacting with AMA of PBC is important to warrant further study of AMA and the cause of the disease. The isolation of responsible antigens had been difficult because the four antigens were insoluble. However, the antigen newly found by us, the 36 kDa fragment, obtained by partial trypsin digestion, is soluble. Using several procedures, the antigenic protein target of AMA was purified from mitochondria for the first time. We determined the N‐terminal sequence of the soluble 36 kDa fragment, 25 residues in length. Until now the N‐terminal sequence of the 36 kDa protein has not shown significant homology with any known protein. The present results of antigen purification would contribute to the elucidation of the epitopes of AMA antigen.
Antigen‐specific electrophoretic cell separation for immunological investigations
Authors: Hansen Ernil , Wustrow Thomas P. U. , Hannig Kurt
Published: May 2005

Preincubation of human blood lymphocytes with cell surface antigen specific antibodies under non‐capping conditions reduces the electrophoretic mobility of the corresponding lymphocyte subpopulation. Antigen‐positive and antigen‐negative cells can be separated by free flow electrophoresis with high yield, purity and viability. The use of fluorescence‐labelled second antibodies augments the induced decrease in net surface charge density, and allows rapid detection of antigen‐positive cells in the fractions of electrophoresis. Carrier‐free cell electrophoresis of human peripheral blood lymphocytes after reaction with anti‐IgM‐antibody or the monoclonal antibodies OKT4 or OKT8, and sandwich staining with tetrarhodamine isothiocyanate‐labelled anti‐IgG resulted in the large‐scale separation of highly pure human B and T lymphocyte subpopulations. Their functional integrity was shown in assays of lymphocyte transformation and of antigen‐specific induction and regulation of antibody synthesis in vitro. These separated lymphocyte subpopulations are useful tools for immunological investigations. While, for instance, the effects of drugs on human lymphocytes are obscured by coincident changes in cell composition of the peripheral blood tested that do not by themselves reflect whole body immunocompetence, the cell separation and in vitro assays at a defined cell number and cell composition allow the recording of quantitative changes in the function of different cell sub‐populations. We studied the influence of the anesthetic thiopental on separated, human lymphocyte subsets. In both polyclonal lectin stimulation and in vitro antibody production, thiopental exhibited a noncytotoxic suppression of lymphocyte functions. B‐Cells, T‐helper and T‐suppressor cells were equally affected and showed the same dose response. The electrophoretic separation of cells after reaction with antibodies is applicable to a wide range of human lymphocyte subpopulations defined by specific antibodies. Their gentle isolation in large quantities is a powerful tool for investigations of the immune system.
Polymorphism of aldehyde dehydrogenase and its application to alcoholism
Authors: Harada Shoji
Published: May 2005

Aldehyde dehydrogenase (ALDH) consists of four different isozymes (I, II, III, IV). Among these, ALDH I shows genetic polymorphism (normal and deficient) in a population of Mongoloid origin. The significantly lower frequency of ALDH I deficiency was found in alcoholic patients compared with healthy controls. ALDH I deficiency is one of the important genetic factors in regulating alcohol consumption and plays a protective role against alcoholism. Population genetic studies on ALDH I deficiency in different ethnic groups indicate that ALDH deficiency was found only in Mongoloid population groups.
Characterization of bacterial genospecies by computer‐assisted statistical analysis of enzyme electrophoretic data
Authors: Picard Bertrand , Goullet Philippe , Bouvet Philippe J. M. , Decoux Gerard , Denis Jean‐Baptist
Published: May 2005

A computer‐assisted statistical treatment of the electrophoretic data obtained from the analysis of two dehydrogenases and 27 kinds of esterases produced by strains belonging to the taxonomically complex genus Acinetobacter is described. The 12 genospecies were clearly separated from each other by correspondence analysis, For each genospecies the distances of the strains from their barycenter were computed and typical isolates suitable for use as reference strains were determined. This approach is suitable for the systematic study of other procaryotic or eucaryotic organisms.
Electrophoresis in one buffer at two pH values
Authors: Michov Budin M.
Published: May 2005

A theory for discontinuous electrophoresis in a polyacrylamide gel is presented for one buffer at two pH values. It is shown that polyions stack between identical leading and trailing ions, and resolve in a gel of constant polyacrylamide concentration. The theory is illustrated by the separation of serum protein polyions in a Tris‐glycinate buffer of pH 8.19 in the well‐forming gel, and pH 9.16 in the resolving gel. The selected concentrations and electrolyte ionization degrees of Tris and glycine have values at which the serum protein polyions stack between the resolving and electrode buffers, followed by separation in the resolving gel.
Determination of the electrophoretic mobility of chromosomes by free flow electrophoresis: I. Morphology and stability
Authors: Bier Frank F. , Bettag Ulrich , Rheingans Thomas , Adrian Hedwig , Barths Jochen , Hausmann Michael , Bühring Hans‐Jörg , Rohwer Peter , Dölle Jürgen , Cremer Christoph
Published: May 2005

Isolated metaphase chromosomes of several fibroblastoid cell lines (Chinese hamster, Chinese hamster × human hybrid) were subjected to free flow electrophoresis (FFE) to study their electrophoretic mobility (EM). The morphology and stability of the chromosomes were unaffected by FFE as examined by cytogenetic methods and flow cytometry. The chromosomes of the complement all showed similar EM under most of the conditions applied. At neutral pH the EM of the chromosomes had the same sign as free DNA and about 2/3 of its magnitude. The variation of EM with buffer parameters such as ionic strength, valence of counterions, buffer capacity and dielectric constant of the solvent were investigated. Thermal denaturation increased the EM of the chromosomes by 20 %. Partial denaturation might offer a possibility to separate or enrich large amounts of chromosomes by FFE.
Gel electrophoresis of native actin and the actin‐deoxyribonuclease I complex
Authors: Edgar Alasdair J.
Published: May 2005

Electrophoresis of monomeric actin (G‐actin) on 8–25 % acrylamide Pharmacia PhastGels was carried out using gels and agarose buffer strips preequilibrated in buffer containing adenosine triphosphate (ATP), calcium ions (Ca2+) and dithiothreitol. On these gels G‐actin ran as a sharp band at an apparent molecular mass of 45 kDa relative to standard proteins which is slightly greater than its actual molecular mass of 42 kDa. Electrophoresis in the absence of these solutes led to denaturation and aggregation of the protein, as reflected by a long streak. Filamentous actin (F‐actin) did not enter the gel. The actin monomer‐binding protein, deoxyribonuclease I, (DNase I) forms a binary complex with G‐actin. The purity and apparent molecular mass 74 kDa of this complex were determined by native gel electrophoresis. By the simple procedure of preequilibrating both gel and buffer strips with appropriate ligands, this technique could be extended to investigate interactions between actin and other G‐actin‐binding proteins and other proteins whose stability is ligand dependent.
Rapid and sensitive silver‐lipopolysaccharide staining using PhastSystem in fast horizontal polyacrylamide gel electrophoresis
Authors: Lee Kuo‐Kau , Ellis Anthony E.
Published: May 2005

A rapid and sensitive silver‐lipopolysaccharide staining method has been developed by using PhastSystem. The total time of the procedure (including time of Phastgel electrophoresis) is within 2 h. It is at least 10 times faster than the previous reported methods and the sensitivity is also increased.
The use of PhastSystem crossed immunoelectrophoresis with immunoblotting to demonstrate a complex between glycoprotein Ib and the actin‐binding protein (ABP) of human platelets
Authors: Aakhus Anne‐Margrethe , Wilkinson Michael , Pedersen Turid Margrethe , Solum Nils Olav
Published: May 2005

The study shows how a technique described in an accompanying paper can be applied to solve a biological problem. The technique makes use of the observation that a monoclonal antibody that has been coprecipitated with its antigen during crossed immunoelectrophoresis can be transferred to a nitrocellulose membrane and visualized. Previous studies using crossed immunoelectrophoresis of Triton X‐100 extracts of platelets have indicated that a particular immunoprecipitate (peak III) of the membrane receptor glycoprotein Ib (GP Ib) might contain a complex between the receptor and the actin‐binding protein (filamin). When a monoclonal antibody (PM6/ 317) directed towards the actin‐binding protein was added to a platelet extract prior to immunoelectrophoresis and blotting, this was visualized on the blot as a replica of the peak III immunoprecipitate. This demonstrates a colocalization of GP Ib and the actin‐binding protein in the precipitate, and thus the existence of a complex between the membrane receptor and the cytoskeletal protein.
Polyacrylamide gradient gel electrophoresis of cytosolic aretinol‐ and retinoic acid‐binding proteins: Application to rat testis and liver
Authors: Melin Anne‐Marie , Carbonneau Marie‐Annette , Maviel Marie‐Josée , Clerc Michel
Published: May 2005

Distribution and cellular levels of retinol‐binding protein and retinoic acid‐binding protein, involved in the molecular action of retinoids, were analyzed in rat testis and liver. Both binding proteins of cytosolic extracts were separated by linear‐polyacryl‐amide gradient gel electrophoresis and following electrophoretic separation, could be visualized by complementary identification tests such as autoradiography and marker proteins. The concentrations of the binding proteins were evaluated by scanning the polyacrylamide gradient gels and the resulting data were found to be in accordance with those obtained by counting radioactivities. Polyacrylamide gradient gel electrophoresis appears suitable to detect and quantitatively evaluate cytosolic retinol‐ and retinoic acid‐binding proteins.
Two‐dimensional electrophoresis of plasma proteins and high density lipoproteins during inflammation
Authors: Choukaife Ala , Visvikis Sophia , Steinmetz Josiane , Galteau Marie‐Madeleine , Kabbaj Ouafae , Férard Georges , Métais Pierre , Siest Gérard
Published: May 2005

Plasma protein and lipoprotein fractions of five patients were analyzed on day 1, 5, and 15 after severe head injury by combining three types of two‐dimensional electrophoresis (2‐DE) to obtain information on lipoprotein and apolipoprotein composition. On analysis under nondenaturing conditions in both dimensions on day 5, the samples show modifications of isoelectric point (pI) and molecular weight (Mr) properties of the high density lipoprotein (HDL) fraction in addition to an increase in inflammatory proteins and a return to a normal pattern on day 15. In the second type of 2‐DE the samples were analyzed employing isoelectric focusing without denaturant in the first dimension, followed by sodium dodecyl sulfate (SDS) in the second dimension in order to study the protein composition of lipoprotein fractions. On day 5, a decrease of the apolipoproteins apo A‐I, apo A‐II, and apo C were noted, with simultaneous appearance of an unidentified protein with Mr 12 000 and pI 6.0. In the third type of 2‐DE, employing urea and Nonidet P‐40 in the first and SDS in the second dimension, the plasma polypeptide composition was studied. The presence of an unidentified polypeptide could be confirmed on day 5, tending to disappear there‐after. This Mr 12 000 component consists of two major spots at pI 5.7 and 6.0 and four minor ones between pI 6.0 and 8.0. These properties suggest that this protein corresponds to serum amyloid A apolipoprotein.
Analysis of virus protein heterogeneity among group B coxsackie viruses using a “mini” two‐dimensional gel electrophoresis system
Authors: Cash Phillip
Published: May 2005

The application of a small format two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) system to the study of protein heterogeneity among group B coxsackie virus (CVB) isolates is described. Under the conditions of electrophoresis developed during this study, protein samples could be processed within 7 h and up to 300 intracellular proteins were resolved from uninfected HEp‐2 cell lysates. 2D‐PAGE was used to characterise the intracellular proteins of clinical CVB isolates of serotypes 4 and 5. Intracellular proteins from virus‐infected cells were radiolabelled using a pulse‐chase protocol under conditions which promoted inhibition of cellular protein synthesis. Depending on the CVB serotype up to 11 intracellular virus proteins were identified, ranging in molecular weight between 14 000 and 54 000. Although the overall two‐dimensional protein profiles were characteristic for the two CVB serotypes, within a CVB serotype there was some heterogeneity of the virus proteins, mainly affecting the proteins' net charge. The sensitivity of 2D‐PAGE in detecting subtle differences in virus proteins combined with the convenience of the small gel format makes this a suitable approach for the study of the molecular epidemiology of human virus pathogens.
Thin‐layer electrophoretic separation of monosaccharides, oligosaccharides and related compounds on reverse phase silica gel
Authors: Scherz Heimo
Published: May 2005

The electrophoretic mobilities of monosaccharides, oligosaccharides, sugar alcoho and sugar acids were determined in 0.3 M borate buffer, pH 10, using thin‐layer electrophoresis on silanized silica gel, pretreated with octanol‐1. A rapid separation of number of sugars, occurring in foods, could be achieved. Using a 0.05–0.1 M neutra solution of barium acetate as electrolyte, thin‐layer electrophoresis allowed exceller and rapid separation as well as identification of all common uronic acids which are constituents of many acidic polysaccharides.
Isolation and recovery of acidic oligosaccharides from polyacrylamide gels by semi‐dry electrotransfer
Authors: Al‐Hakim Ali , Linhardt Robert J.
Published: May 2005

Acidic oligosaccharides derived from glycosaminoglycan heparin were separated by polyacrylamide gradient gel electrophoresis (PAGE). The gel could be visualized using Alcian Blue dye to give a pattern of highly resolved, well defined bands. The particular banding pattern obtained was the result of a heparinase catalyzed depolymerization which afforded oligosaccharide products that differed in size by one disaccharide unit. The separated oligosaccharides could be recovered prior to staining by electroelution onto a positively charged nylon membrane by a semi‐dry transfer procedure. Subsequent elution and quantitative recovery of individual oligosaccharides from the membrane was achieved. By using multiple membrane layers a second separation dimension was obtained, resulting in increased oligosaccharide purity proportional to transfer depth. Preparative gradient polyacrylamide gel electrophoresis followed by semi‐dry electro‐transfer and recovery represents a novel method for the preparation of homogeneous acidic oligosaccharides.