bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple successive immunoprinting: A fast blotting technique of a single agarose isoelectric focusing gel
Authors: Desvaux François X. , David Bernard , Peltre Gabriel
Published: May 2005

Multiple successive pressure blottings of a single agarose isoelectric focusing gel were performed on normal and CNBr‐activated nitrocellulose (NC) filters. The results obtained by multiple successive 10s immunoprints were compared to those obtained by a single 10 min immunoprint. To quantify the transfer efficiency of these techniques, a defined amount of radioactive material was separated by isoelectric focusing on agarose gel. After separation and pressure blottings of the gel the NC filters were submitted to autoradiography. The amount of radioactive material bound to the NC filters was determined by scintillation technique. The single 10 min pressure blot was more efficient than each of the multiple successive 10s prints. However, the latter procedure allowed equal resolution and resulted in a higher recovery of total radioactivity than the single immunoprint technique. The aim of this paper is to show how to obtain highly reproducible prints of electrophoretic patterns in an agarose gel of heterogeneous samples when accurate multiple immunodetections are to be performed. This technique was tested to characterize the grass pollen specific immunoglobulin classes and subclasses from an allergic patient.
Analysis of plasma membrane protein changes in Dictyostelium discoideum during concanavalin A induced receptor redistribution using two‐dimensional gel electrophoresis
Authors: Patton Wayne F. , Dhanak Michelle R. , Jacobson Bruce S.
Published: May 2005

The 127 major polypeptides obtained from the purified plasma membrane of Dictyostelium discoideum were examined using two‐dimensional gel electrophoresis and a microcomputer‐based videodensitometer. Plasma membrane proteins were analyzed at four discrete stages of concanavalin A induced cell surface capping; (i) the cell surface in the absence of ligand (unbound), (ii) the surface immediately after ligand binding (bound), (iii) the cell surface after receptors had patched (patched) and (iv) the cell surface after receptors had capped (capped). Plasma membranes were obtained at various stages of capping by using a colloidal silica density perturbation technique which immediately immobilized the proteins, preserving their lateral distribution in the bilayer during the isolation. Proteins were characterized with respect to post‐translational modification changes resulting from the capping process as well as changes in their association with the plasma membrane fraction. Posttranslational changes of plasma membrane proteins, such as phosphorylation, methylation and proteolytic cleavage, were not observed during the four stages of capping. Myosin heavy chain phosphorylation, however, decreased almost twofold during patching and capping. Actin, which is known to colocalize directly underneath capped receptors did not appear to be recruited to the cap from the cytoplasm.
Separation of some triazine herbicides and their solvolytic products by capillary zone electrophoresis
Authors: Foret František , Šustáček Vladimír , Boček Petr
Published: May 2005

The separation of triazine herbicides and their solvolytic products by capillary zone electrophoresis in mixed water‐ethanol background electrolytes is described, allowing the analysis of picomole amounts of a triazine mixture in less than 15 min.
Electrophoretic purification of the alpha and beta subunits of phosphorylase kinase and evidence in support of the deduced amino acid sequences
Authors: Crabb John W. , Harris William R. , Johnson Charles M. , Sotiroudis Theodore G. , Kuhn Carl C. , Heilmeyer Ludwig M. G.
Published: May 2005

A simple, rapid sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) method is presented for isolating the α, α' and β subunits of rabbit muscle phosphorylase kinase. The SDS‐PAGE procedure can yield milligram amounts of α and β from a single preparative gel and also allows isolation of the α' isozyme free of α. Notably the method provides the purified subunits in a form amenable to structural analysis. Edman degradation of α and α' reveal identical NH2‐terminal structures. Amino acid analysis of the electrophoretically purified α and β subunits are in good agreement with their deduced primary structures. The amino acid sequence of 488 residues in α and 713 residues in β were determined by gas phase Edman degradation. The data support the recently deduced primary structures of α (Zander et al., Proc. Natl. Acad. Sci. USA 1988, 85, 2929–2933) and of β (Kilimann et al., Proc. Natl. Acad. Sci. USA, 1988, 85, 9381–9385).
IPGMAKER: A program for IBM‐compatible personal computers to create and test recipes for immobilized pH gradients
Authors: Altland Klaus
Published: May 2005

The program “IPGMAKER” is a computational aid for creating and testing recipes for near‐linear immobilized pH gradients. It was written for fast IBM personal computers (with a Type 80386 processor and 80387 coprocessor) and compatibles equipped with a VGA, EGA or Hercules (mono) graphics card. The program is limited to the use of up to 10 acids and/or bases, and to ranges spanning between pH 2 and 12. The resulting recipes are presented either as final concentrations in the 2 chambers of a mixing device for linear gradients or as volumes from 0.2 moles/L stock solutions adjusted to a user‐defined average buffering power. One of the subroutines determines the pH, gradient slope and buffering capacity at any location of the gradient and includes a facility to estimate the pI of proteins from the composition of their primary structure.
Fabric‐reinforced ultrathin polyacrylamide gels for sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and isoelectric focusing
Authors: Peisker Klaus
Published: May 2005

The suitability of four different fabric materials for the preparation of ultrathin reinforced polyacrylamide gels was investigated. With all fabric‐reinforced gels, a good separation of proteins by isoelectric focusing and sodium dodecyl sulfate‐electrophoresis could be achieved. Semi‐dry electrophoretic blotting of proteins was possible with all types of fabric‐reinforced gels. Two polyester fabrics (a net and a fleece) were decidedly superior in handling and dimensional stability on drying to a nylon fabric and another polyester fleece material. Only gels prepared with the former materials withstood further treatment, such as fixation, staining, destaining, and drying. One of the polyester fleece fabrics had poor handling properties and the nylon fabric was unsuitable for direct staining procedures employing concentrated (20 % w/v) trichloroacetic acid as fixative.
Degradation of individual intracellular proteins analyzed by two‐dimensional gel electrophoresis and computerized video densitometry
Authors: Rodemann H. Peter
Published: May 2005

A technique is described for the analysis of degradation rates of individual intracellular proteins, based on pulse‐chase‐labeling of cells using radioactive amino acids [35S]methionine, two‐dimensional polyacrylamide gel electrophoresis, fluorography and scanning of the fluorograms by a computerized video densitomter. As compared to scintillation counting of individual protein spots resolved by two‐dimensional gel electrophoresis, this method allows a rapid and precise determination of the degradation rates of individual intracellular proteins. In the present study, degradation rates of individual intracellular proteins of normal human skin fibroblasts and skin fibroblasts from patients with Duchenne muscular dystrophy were compared. Rates of degradation for proteins PIIa, PIIb and PIIc recently described as cell‐type‐specific proteins were significantly enhanced (p > 0.01) in fibroblast cultures of Duchenne muscular dystrophy origin.
Electrophoresis in an expanded stationary boundary
Authors: Michov Budin M.
Published: May 2005

A method of electrophoresis in an expanded stationary boundary is described, in which a pH gradient formed in a single buffer is used. The degrees of ionization and the concentrations of the electrolytes have values at which the polyions stack and resolve simultaneously in a polyacrylamide gel. To illustrate the method, the resolving of serum proteins in a pH gradient formed by Tris – glycinate buffer is considered.
Free flow electrophoresis for the purification of proteins: I. Zone electrophoresis and isotachophoresis
Authors: Hoffstetter‐Kuhn Sabrina , Kuhn Reinhard , Wagner Horst
Published: May 2005

The principles and some applications of free flow zone electrophoresis and isotachophoresis are described. The influence of (i) carrier electrolyte conductivity on the migration velocity and (ii) band shape on zone electrophoresis was investigated. The technique was found convenient for studying the effect of pH on the mobility of proteins to create a mobility curve. The purification of alcohol dehydrogenase from a crude yeast extract revealed the separation power of zone electrophoresis for complex protein mixtures. Without additional steps, a purification factor of 5.4, with a recovery of 97 % alcohol dehydrogenase, was achieved. Free flow isotachophoresis was applied to the purification of immunoglobulins from human serum. Disadvantages of this technique are the time‐consuming development of an optimized separation system and the empirical search for suitable spacers. Also, reaching of the steady state becomes increasingly difficult as the number of sample components increases.
Electrophoresis of gliadins in long acrylamide gels: Method and nomenclature
Authors: Branlard Gérard , Picard Brigitte , Courvoisier Christian
Published: May 2005

Gliadins, defined as wheat kernel storage proteins soluble in 70% ethanol, possess an electrophoretic diversity permitting us to identify different varieties. Because of genetic proximity, however, it sometimes proves impossible to distinguish between different varieties using standard methods. An electrophoresis method, utilizing a discontinuous buffer system with aluminum lactate and potassium lactate in the gel and sodium lactate in the electrode vessels, is described for two types of gels 18 cm and 32 cm in length. The number of bands in higher than with standard methods, and is significantly increased with long gels, facilitating the distinction between varieties. A nomenclature for the new bands is presented for several widely different cultivars.
Haptoglobin subtyping by isoelectric focusing in miniaturized polyacrylamide gels rehydrated in presence of 2‐mercaptoethanol
Authors: Alonso Antonio , Visedo Guillermo , Sancho Manuel , Fernández‐Piqueras José
Published: May 2005

A fast isoelectric focusing method for routine haptoglobin (Hp) subtyping is presented. This method is based on isoelectric focusing, under reducting conditions, of neuraminidase‐treated plasma samples by using dry miniaturized (interelectrode distance: 55 mm) polyacrylamide gel, rehydráted in presence of 2‐mercaptoethanol and a mixture of pharmalyte carrier ampholytes (pH 4–6.5 and pH 6–8) followed by immunoblotting. The presence of 2‐mercaptoethanol in the gel prevented refolding of the Hp α and Hp β chains during focusing, making it possible to obtain a sharp Hp band pattern with a clear separation of the different Hp α allelic products (1S, 1F, 2FS, 2SS and 2FF). A population study carried out with 250 unrelated individuals living in Central Spain is also presented.
Human skeletal muscle myosin light chains analyzed by immobilized pH gradients during ontogenesis: Identification of new phosphorylatable isoforms of light chain 2
Authors: Pernelle Jean‐Jacques , Righetti Pier Giorgio , Wahrmann Juan P. , Herve Bernadette
Published: May 2005

Previous studies using two‐dimensional gel electrophoresis have described adult and fetal isoforms of skeletal muscle myosin light chains (MLC). They have also revealed an embryo‐specific light chain (LC1emb), apparently absent in most adult skeletal muscles. In order to characterize more thoroughly the MLC family, we have analyzed the MLCs from human skeletal muscle at different developmental stages using a two‐dimensional electrophoresis technique with an immobilized pH gradient in the first dimension. The high resolution of this novel technique, resolving components which in isoelectric points are ≤ 0.01 pH, combined with sensitive silver staining, has allowed us to identify four phosphorylatable isoforms of MLC2: two slow‐myosin light chains (MLC2Sa and b), two fast myosin light chains (MLC2:a and b), and their phosphorylated counterparts: MLC2SaP and bP, MLC2FaP and bP. The following major modifications during development were observed: (i) The embryonic LC (LC1 emb) persists up to at least 26 weeks of fetal life. (ii) The polymorphism of LC2 is already evident at 10 weeks of development but only the nonphosphorylated forms of LC2S and LC2F seem to be present. The LC2Fa form is predominant. As early as 26 weeks of fetal life, the 4 phosphorylated forms are detected. In the adult, LC2Fb is a minor component. (iii) LC3F (fast) is already expressed at an early embryonic stage (10 weeks).
Abnormal protein spots revealed by two‐dimensional electrophoresis in mycoplasma‐infected human fibroblasts
Authors: Yang Rei‐Cheng , Tsuji Akihiko , Suzuki Yoshiyuki
Published: May 2005

Six new protein spots were detected by two‐dimensional electrophoresis in cultured human fibroblasts infected with mycoplasma. No changes were observed in other protein spots present in normal and pathological cells. After treatment with an antimycoplasma drug for a week, the new spots disappeared and the cells became negative for mycoplasma stain.
Autoradiography using storage phosphor technology
Authors: Johnston Richard F. , Pickett Siobhan C. , Barker David L.
Published: May 2005

We describe the application of photostimulable storage phosphor imaging plates to autoradiography of samples labeled with 32P, 14C and 35S. Imaging plates can detect a 32P‐labeled sample at an exposure level of 1 disintegration/mm2, and 14C and 35S at 25 disintegrations/mm2. Compared to X‐ray film, imaging plates offer increased sensitivity of 15‐ to 250‐fold for 32P and 20‐ to 100‐fold for 14C and 35S. Resolution is in the 0.3 mm range and is therefore suitable for any gel or blot application.
The application of schlieren optics for detection of protein bands and other phenomena in polyacrylamide gel electrophoresis
Authors: Takagi Toshio , Kubota Hidehiro
Published: May 2005

A modernized schlieren optics system was applied to follow protein bands visually during polyacrylamide gel electrophoresis. This approach is suitable for quantitative measurement of electrophoresis as well as for detection of protein bands because of the absence of distortion of the electrophoretic pattern during the staining process. The optics are also suitable for the detection of various background phenomena which usually escape observation.
Quantitative autoradiographic imaging using gas‐counter technology
Authors: Bateman J. Edmond
Published: May 2005

Gas counters based on the multiwire proportional counter (MWPC) and multistep avalanche (MSA) technologies can combine the functions of imaging and quantitation (or assay) of radioactively labelled electrophoretograms and blots. Submillimeter spatial resolutions combined with good quantitation have been achieved for the most common beta‐emitting radio labels. The current state of development of this technology is reviewed and assessments of the performance of these devices relative to the more traditional systems (X‐ray film and the scintillation counter) presented. Particular attention is paid to the potential of these devices to enhance the productivity of the scanning of blots and the performance of assays.
Separation of durum wheat proteins by ultrathin‐layer isoelectric focusing: A new tool for the characterization and quantification of low molecular weight glutenins
Authors: Morel Marie‐Hélène , Autran Jean‐Claude
Published: May 2005

An isoelectric focusing method capable of resolving all groups of storage protein of the wheat seed, including the most basic low molecular weight glutenin (LMWG), was developed. Ultrathin polyacrylamide gels were used after drying and rehydration with 8 m urea, 50 mm DTE and 2.4 % carrier ampholytes (pH 4–9). Densitometric scanning of the isoelectric focusing gels permitted a more accurate and specific quantitation of LMWG components among various cultivars than patterns based on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The two main genetic types (i. e. ‘γ‐42’ and ‘γ‐45’) of durum wheats were separated on the basis of the proportion in LMWG in storage proteins, but no significant difference was found within these groups. Advantages of the system as regards reliability, high resolution ability to abolish protein oxidation and preventing reaggregation of LMWG were also discussed.
Quantitative densitometry from the point of view of information theory
Authors: Pardowitz Iancu , Ehrhardt Wolfgang , Neuhoff Volker
Published: May 2005

The process of quantitative densitometry is analyzed with methods developed in information theory. It is shown that the steps involved in densitometry, e. g. gel staining, mechanical, optical and electronic processing, as well as all the steps of data processing, can be viewed as communication channels. The factors affecting both the relevant and irrelevant part of the total information passed through these channels are discussed in the consistent frame provided by information theory. This view leads to a unifying context for analyzing the performance of quantitative densitometers.
GELANAL, a personal computer‐program to compare protein patterns on two‐dimensional polyacrylamide gels
Authors: Klerk Hans , Jespers Ad
Published: May 2005

Comparing and analyzing a series of two‐dimensional gels by hand is troublesome and subjective. So far a number of systems for automatic analysis have been developed on mainly mainframe computers, using complex algorithms. This paper presents an inexpensive system, based on a simple Pascal program, to compare individual spots on two‐dimensional gels using an IBM or compatible personal computer in a qualitative way. The accuracy of the method is demonstrated by comparing two patterns of the same extract from different runs.
Haptoglobin subtyping with anti‐haptoglobin α chain antibodies
Authors: Shindo Shoko
Published: May 2005

Specific antibodies against the human haptoglobin (HP) α chain were raised and used for immunoblotting after isoelectric focusing to determine Hp α subtypes in a reproducible and simplified manner. By eliminating the staining of the HP β chain, HP α subtypes were visualized more precisely and simply than by previously reported methods. Subtypes of a total of 1211 sera, obtained from two different populations in Japan, were examined by the new method. Four HP α subtypes (HP 1S–1S, 2FS–1S, 2FS–2FS and 2FS–2SS) and five subtypes combined with a new variant (HP 2FS–1V1, 2V1–1S, 2FS–2V1, 2FS–2V2 and 2FS–2V3) were observed. HP 1V1 belonged to HP α1, and HP 2V1, 2V2 and 2V3 belonged to HP α2, and their alleles were designated HP A*1V1, HP A*2V1, HP A*2V2 and HP A*2V3, respectively. The allele frequencies were calculated to be as follows: HP A*1S, 0.2597; HP A*1V1, 0.0004; HP A*2FS, 0.7333; HP A*2SS, 0.0008; HP A*2V1, 0.0045; HP A*2V2, 0.0008; and HP A*2V3, 0.0004. The allele frequency of HP A*2SS, which is common in the European population, is less frequent than HP A*2V1 in the Japanese population.
Zymographic screening of plant peroxidase isoenzymes oxidizing 4‐hydroxystilbenes
Authors: Calderón Antonio A. , Pedreño M. Angeles , Ros‐Barceló Alfonso , Muñoz Romualdo
Published: May 2005

A zymographic assay is described for the detection of peroxidase isoenzymes oxidizing 4‐hydroxystilbene following isoelectric focusing. The assay is based on coupling intermediate products of the oxidation of 4‐hydroxystilbene with 4‐aminoantipyrine, with resultant formation of dye complexes. Control experiments in the absence of 4‐hydroxystilbene and hydrogen peroxide demonstrate the peroxidative nature of the 4‐hydroxystilbene‐dependent dye reaction.
Microsequencing of proteins electrotransferred onto immobilizing matrices from polyacrylamide gel electrophoresis: Application to an insoluble protein
Authors: Hirano Hisashi , Watanabe Toshihiro
Published: May 2005

Semidry electroblotting is convenient and allows a rapid and efficient protein transfer from one‐ or two‐dimensional polyacrylamide gels onto sequencer stable supports for protein microsequence analysis in a gas‐phase sequencer. Using this technique, we determined the amino acid sequences of the basic 7S globulin (Bg), an insoluble protein present in soybean seeds. Based on sequence determination, the cDNA‐encoding Bg could be easily cloned and characterized.
Calibration of polyacrylamide gel columns for the separation of oligonucleotides by capillary electrophoresis
Authors: Paulus Aran , Gassmann Ernst , Field Matthew J.
Published: May 2005

Polyacrylamide‐filled gel columns are used to separate oligonucleotide samples. For homopolymeric standard samples, plots of migration time versus molecular size are presented over a range of 30–160 bases. With 2.5–4 % T and 3.3 % C gels, good resolution over the examined mass range, with peak width at half height of 3 to 6 s, is obtained by applying electrical fields of 200–400 V/cm. The separation of heteropolymeric nucleotides by slab gel electrophoresis under routine conditions was compared with capillary gel electrophoresis. Using the same column and the same separation conditions, the plot of migration time versus base number is linear with and identical slope for three oligonucleotide samples which were examined, allowing a calibration of a gel‐filled capillary for molecular mass determination.
Capillary zone electrophoresis separation and laser‐based detection of both fluorescein thiohydantoin and dimethylaminoazobenzene thiohydantoin derivatives of amino acids
Authors: Waldron Karen C. , Wu Shaole , Earle Colin W. , Harke Heather R. , Dovichi Norman J.
Published: May 2005

Capillary zone electrophoresis is employed for the separation and analysis of both fluorescein thiohydantoin and dimethylaminoazobenzene thiohydantoin derivatives of amino acids. Detection of minute amounts of these amino acid derivatives is an important milestone in the development of a high sensitivity protein sequencer. Current detection limits for the fluorescein derivative is on the order of 10−21 moles whereas detection limits for the dimethylaminoazobenzene derivative is on the order of 10−16 moles.
Multiphasic electrophoresis with diversified spacers
Authors: Bringard Aline , Charlionet Roland
Published: May 2005

The principle and some applications of multiphasic electrophoresis with diversified spacers are described. The method combines previously published concepts of introducing highly diversified spacers into the stack of an isotachophoretic system with the theory of snow‐ and telescope‐electrophoresis. The link between this electrophoretic method and isotachophoresis on the one hand and monophasic zone electrophoresis on the other hand is exemplified. The importance of the nature of the spacers as well as of the pH and ions in the leading and terminating zone is illustrated. Details are given concerning the experimental set‐up. This electrophoretic technique could serve as an alternative to isoelectric focusing.
Isoelectric focusing in immobilized pH gradients of phosphoglucomutase and esterases from the spiny lobster
Authors: Espinosa Georgina , Wenisch Elisabeth , Danner Herbert , Katinger Hermann , Righetti Pier Giorgio
Published: May 2005

A method is described for detecting polymorphisms of cephalothorax and tail homegenates of 25 puerulus staged Panulirus argus in phosphoglucomutase (PGM) a esterases. Isoelectric focusing in immobilized pH gradients was used. In the pH 6.0–8.0 interval for phosphoglucomutase and in the pH 3.5–5.0 and 4.2–4.9 rang for esterases both enzymes appeared as polymorphic band patterns. These could explained by one locus with 2 alleles for phosphoglucomutase and 3 loci with 2, 3 and 4 alleles for esterases. Esterases exhibit a more extensive polymorphism in immobilied pH gradients than in polyacrylamide gel electrophoresis.
Hereditary subtypic patterns detected in the Ba fragment of complement factor B: Occurrence of four common alleles in Japanese
Authors: Suzuki Koichi , Ito Shigenori , Tamura Akiyoshi , Fujita Kiyoshi , Matsumoto Hideo
Published: May 2005

A procedure which can detect subtype‐specific minor bands of factor B (BF) by polyacrylamide gel isoelectric focusing is presented. After zymosan‐mediated fragmentation of BF in serum via alternative pathway for complement activation, serum samples are subjected to isoelectric focusing in a narrow pH range (4.2–4.9). The Ba fragments are detected by using immunoblotting. In addition to the previously reported minor bands with subtypic specificities, heterogeneities are observed in other minor band group, where a single minor band corresponds exclusively to a subtype in a regular combination with the previously announced subtypic patterns. A one‐to‐one correspondence of a single band to each subtype provides an unambiguous determination for three subtypic phenotypes deduced from the two divided BF*F alleles, BF*FA and BF*FB. An autosomal codominant heredity is confirmed through segregation analysis. A population survey reveals that four common alleles, BF*S, BF*FA, BF*FB, BF*Fb1, occur in a Japanese population and the former three alleles, except BF*Fb1, occur in a Cambodian population. The presence or absence of a single anodal minor band was found to be the only difference after neuraminidase treatment of FA and FB, implying that an amino acid substitution responsible for the FA–FB subtypic difference is involved in an additional acquisition in FA of an oligosaccharide unit with a charged sialic acid.
Analysis of caprine mycoplasmas and mycoplasma infections in goats using two‐dimensional electrophoresis and immunoblotting
Authors: Olsson Birgitta , Bölske Göran , Bergström Katrin , Johansson Karl‐Erik
Published: May 2005

Seven stains of mycoplasma have been characterized by two‐dimensional polyacrylamide gel electrophoresis in combination with immunoblotting. The two‐dimensional patterns of the strains were compared and similarities and differences are discussed. Hyperimmune rabbit antisera were used in the immunoblotting experiment to study the immunological cross‐reactivity between the species. Sera from goats, naturally and experimentally infected with mycoplasma, were also used in the immunoblotting experiment to study the immune responses of the infected animals.
Two‐dimensional electrophoresis of the fatty acid binding protein from human heart: Evidence for a thiol group which can form an intermolecular disulfide bond
Authors: Nielsen Søren U. , Vorum Henrik , Spener Friedrich , Brodersen Rolf
Published: May 2005

A 100 000 g supernatant from human heart muscle, containing cytosolic proteins with some contaminating plasma proteins, was analyzed for fatty acid binding protein (FABP) by two‐dimensional electrophoresis (2‐DE) using isoelectric focusing under nondenaturing conditions in the first dimension. FABP purified from human heart muscle was found to comigrate with a major spot in 2‐DE gels of the supernatant. This spot was comparable with those of the myoglobins in staining intensity. When purified FABP was charged with [H]palmitate and subjected to nondenaturing 2‐DE, radioactivity always comigrated with this protein. Under denaturing and reducing conditions in the second dimension, FABP was found to have a pI of 5.3 and an apparent molecular weight of 15 000. Isoforms of FABP, reported here for the first time to occur in human heart muscle, were observed as minor spots focusing at pH 5.1 and 5.7. When electrophoresis in the second dimension was carried out under denaturing but nonreducing conditions, an additional protein appeared at pH 5.3 with an apparent molecular weight of about 30 000. This protein was identified as a dimer of FABP and evidence for the involvement of an intermolecular disulfide bond in this dimerization is presented.
Electrophoresis of nonreduced IgM and IgG monoclonal antibodies on mixed agarose‐polyacrylamide gels
Authors: Slavíčková Alena
Published: May 2005

A gel matrix composed of 0.5 % agarose and 2.5 % polyacrylamide allows the parallel electrophoretic separation of nonreduced IgM and IgG monoclonal antibodies. The mobilities of IgM and IgG differ at pH 9.5, which enables a preliminary Ig class designation. It allows rapid detection of incomplete cloning if both IgM and IgG are present. A semiquantitative evaluation of nonreduced IgM in ascitic fluid is possible as well.
Numerical technique and computational procedure for isotachophoresis
Authors: Palusinski Olgierd A. , Su Yu , Fife Paul C.
Published: May 2005

This paper presents a new numerical method for computation of solutions of prototypical equations of isotachophoresis. Numerical computation is complicated because the Poisson equation, which relates electrostatic potential to space charge density, contains a small parameter. This parameter is usually assumed to have the value of zero. Under this assumption the Poisson differential equation is replaced by an algebraic equation, which is often called the equation of electroneutrality, because it indeed states that the electrolyte is electrically neutral this assumption were not studied in the past. Here we propose an iterative procedure which allows for computation of solutions without the assumption of electroneutrality. The accuracy is controlled by a number of iterations and is limited by a computer round‐off error only. The method is based on our previously published theory of existence and uniqueness of solutions of isotachophoretic equations. Details of the computational algorithm for prototypical equations of isotachophoresis are given. A numerical example and comparison with previously published data are also provided.
Conditions for purification of proteins by free‐flow zone electrophoresis
Authors: Clifton Michael J. , Jouve Nicolas , Balmann Hélène De , Sanchez Victor
Published: May 2005

The separation of proteins by free‐flow zone electrophoresis is generally impaired by a number of secondary effects which cause spreading of the protein streams with resultant loss in resolution. A strategy is outlined, based on experimental observations and numerical modeling, which allows the operating conditions and separation chamber dimensions to be chosen so as to obtain complete separation of two proteins of known mobility. This approach takes into account some dispersive phenomena such as molecular diffusion, electroosmosis and residence time gradients. In some cases, the right operating conditions cannot be achieved on earth and electrophoresis separations in microgravity may then be justified.
The development of recycle zone electrophoresis
Authors: Ivory Cornelius F.
Published: May 2005

Milan Bier′s contributions to preparative electrophoresis and, in particular, his work in „recycling”︁, have had an enormous impact on the development of scaling strategies for continuous flow electrophoresis. This paper reviews my early work on the theory of recycle applied to zone electrophoresis and reports the results of several recent experiments in which proteins were purified from complex feed mixtures using recycle zone electrophoresis.
Membrane active plasma factor in multiple sclerosis: Characterization and isolation by recycling isoelectric focusing
Authors: Tamblyn Cherry H. , Seaman Geoffrey V. F. , Egen Ned B. , Bier Milan
Published: May 2005

Recycling isoelectric focusing is a rapid, high resolution technique that has the capability of fractionating complex mixtures of proteins on a preparative scale on the basis of their isoelectric points (pIs). For this reason, it appeared to be an ideal tool to further characterize and isolate the surface active plasma component(s) which is abnormal in multiple sclerosis(MS). The normal control and the abnormal MS plasma components, or factors, proved to be stable under the conditions used in this technique, including deionization by electrodialysis, dialysis against distilled water, lyophilization and the presence of 3M urea and carrier distilled water, lyophilization and the presence of 3M urea and carrier ampholytes. The presence or absence of plasma factor activity was determined by incubating red blood cells in a test plasma, or plasma fraction, followed by the determination of the red blood cell electrophoretic mobility in the presence and absence of linoleic acid. Both the normal and MS factor had a pI of 4.0 ± 0.1 under the conditions used. A high degree of purification was achieved and albumin was eliminated as a possible candidate for the factor(s).
Gene‐Protein database of Escherichia coli K ‐ 12: Edition 3
Authors: Vanbogelen Ruth A. , Hutton M. Elizabeth , Neidhardt Frederick C.
Published: May 2005

The first two editions of the E. coli Gene‐Protein Index were published to provide identifications of protein spots resolved by two‐dimensional gel electrophoresis as the products of known genes. This third edition has been expanded to include information about genes and proteins gained directly from two‐dimensional gel analysis – including information about protein spots not yet characterized genetically or biochemically – and is therefore more properly called a cellular proteins database. An alpha‐numeric designation has been uniquely assigned to each of the 616 polypeptide spots in the current database. To this, information is linked about the polypeptide′s identification (protein name, gene name, Enzyme Commission – EC number), location on reference gels (x‐y coordinates), genetics (Genbank code, DNA sequence reference), biochemistry (molecular weight, isoelectric point), and physiology (steady state level of the protein as a function of media and temperature, membership in various regulons and stimulons).
The distribution of particles characterized by size and free mobility within polydisperse populations of protein‐polysaccharide conjugates, determined from two‐dimensional agarose electropherograms
Authors: Tietz Dietmar , Aldroubi Akram , Schneerson Rachel , Unser Michael , Chrambach Andreas
Published: May 2005

New approaches for the characterization of polydisperse particle populations are presented

Preliminary reports of this study were presented at the American Electrophoresis Society Meeting, Washington DC, USA, July 6–11, 1989; at the NIH/ADAMHA‐Industry Collaboration Forum, Bethesda, Maryland, USA, October 3, 1989; at the Electrophoresis Forum '89, Munich Germany, October 23–25, 1989; and at Frontiers in Bioprocessing II, Boulder, Colorado, USA, June 17–21, 1990.

. The investigated samples contain virus‐sized protein‐polysaccharide conjugates which had previously been prepared as immunogens against bacterial meningitis (Hib). The analysis is based on two‐dimensional agarose electrophoresis (Serwer‐type). This method, like the one of O'Farrell, achieves a separation according to size and charge. It relies on a different principle, however, and is applicable to nondenatured particles which are 100 to more than 1000 times larger in mass than regular uncrosslinked proteins. Data from stained gel patterns are evaluated by the computer program ELPHOFIT, which makes it possible to standardize the gel and to construct a nomogram which defines every position on the gel in terms of particle size and free mobility (related to surface net charge density). The output of ELPHOFIT, consisting of nomogram parameters, is transferred to the image processing program GELFIT. This software is used to evaluate the computer images obtained by digitizing the stained gel patterns: (i) The nomogram is electronically superimposed on the computer image. (ii) The gel pattern is transformed from a curvilinear to a rectangular coordinate system of particle size and free mobility. The center of gravity as well as density maxima are given in coordinates of particle size and free mobility. Ranges of grey levels can be accentuated by adding 16 pseudocolors. (iii) Using surface‐stripping techniques, GELFIT provides an estimate for the number of major subpopulations within each preparation. (iv) Numerical values or the distribution of particle size and free mobility are determined. Using program IMAGE, the quantitative physical assessment of a given conjugate preparation is presented in the form of a computer‐generated three‐dimensional plot, the shape of which serves to identify and characterize the preparation visually. The data analysis based on digitized two‐dimensional gel patterns is automated to an extent that a technician can perform routine evaluations. It uses the Macintosh II personal computer.
A rapid and economical method for hybrid purity testing of tomato (Lycopersicon esculentum L.) F1 hybrids using ultrathin‐layer isoelectric focusing of alcohol dehydrogenase variants from seeds
Authors: van den Berg Bartel M.
Published: May 2005

In the cultivated tomato (Lycopersicon esculentum L.), two electrophoretic variants of the enzyme alcohol dehydrogenase (ADH) encoded by the alleles Adh‐1+ and Adh‐11 are found. A rapid and economic method for testing the hybrid purity of tomato F 1 seeds, based on the expected presence of Adh‐1 alleles, was developed. The method is based on the analysis of the ADH variants by ultrathin‐layer isoelectric focusing, pH range 3–10, of crude extracts from imbibed seeds followed by enzyme activity staining. The isoelectric points (pIS) of the ADH variants were estimated to be 5.5 and 5.7 for Adh‐1+ and Adh‐11; respectively. Using the procedure described and a newly developed sample applicator strip, it is Possible for one person to routinely analyze 1152 seeds per day using only a single electrophoresis unit. An investigation of a large number of inbred lines, both experimental and commercial hybrids, together with open‐pollinated varieties, showed the potential of the method. Among F1 hybrids, a higher frequency of the Adh‐11 allele was found than among open pollinated varieties, suggesting that F1 hybrid breeding has resulted in a higher frequency of Adh‐11 alleles by selection of linked genes.
Analysis of two‐dimensional electrophoretic patterns of proteins obtained from the sera of normal and tumorbearing nude rats
Authors: Yoshida Motoko , Itoh Michio , Imai Toshio , Tanimoto Yoshifumi , Sakurabayashi Ikunosuke , Furuya Shin
Published: May 2005

To classify two‐dimensional electrophoresis (2‐DE) patterns of normal and abnormal samples, a new parameter, named shift value, is introduced. The shift value is defined by the product of three indices; differences in density, differences in area, and the Euclid distance between peaks of matched glycoprotein spots in the 2‐DE patterns. Shift values obtained from the differences between the 2‐DE patterns of tumorbearing and normal sera (control) were always found to be larger than those obtained from two control patterns. The shift value obtained from glycoprotein spots could be more effectively used to distinguish normal and abnormal patterns than that obtained from simple proteins. In our earlier attempts to compare serum proteins of normal and tumor patients, we could not discriminate differences caused by genetic background. In order to circumvent this difficulty, we employed an inbred strain of nude rats into which various types of human cancer had been implanted. Reliability of the analysis is enhanced by using a polyacrylamide gel backed with a silanized glass support and an automatic 2‐DE apparatus.
Genome scanning by two‐dimensional DNA typing: The use of repetitive DNA sequences for rapid mapping of genetic traits
Authors: Uitterlinden André G. , Slagboom Eline P. , Mullaart Erick , Meulenbelt Ingrid , Vijg Jan
Published: May 2005

The existence of repetitive DNA sequences offers the possibility to assess the mammalian genome for individual variation in its entirety rather than at one or only a few sites. In order to fully explore the various sets of mammalian repeat sequences for this purpose, analytical tools are required which allow many if not all individual members of sets of repetitive elements to be resolved and identified in terms of location and allelic variation. We have applied and further developed an electrophoretic system, two‐dimensional DNA typing, which may fulfill these requirements. The two‐dimensional system combines separation of DNA fragments is by size in a neutral gel, with separation by sequence composition in a denaturing gradient gel. By hybridization with minisatellite‐ and simple‐sequence core probes and by inter‐repeat polymerase chain reaction techniques, it is possible to obtain individual ‐ and even chromosomespecific separation patterns that consist of hundreds of spots. Computerized image analysis and matching of such spot patterns allow the rapid assesment of multiple polymorphisms, spread over the genome, to monitor genetic variability in populations. When coupled to databases of polymorphic DNA markers with a known genomic location, two‐dimensional DNA typing can greatly accellerate the mapping of genetic traits in humans, animals, and plants.
Highly instable (GATA)n‐containing sequences of the mouse during the cloning process
Authors: Studer Roland , Kammerbauer Claudia , Zischler Hans , Hnkkanen Ari
Published: May 2005

Mouse DNA fingerprints were obtained by HaeIII digestion of genomic DNA and ingel hybridization with the (GATA)4 oligonucleotide probe. In order to obtain locusspecific probes that hybridize with only one fragment of the (GATA)4 DNA fingerprint, a genomic library of size‐selected inserts was constructed using a system of direct subcloning from the phage clones. During the cloning procedure, the phage as well as the plasmid insert DNAs changed primarily within their repetitive DNA but also within adjacent nonrepetitive sequences, as was demonstrated for several clones by in‐gel hybridization with the (GATA)4 probe as well as by sequence analysis. Isolated subclones varied within their (GATA)n repeats, resulting in different insert lengths. Several “metastable” as well as stable (GATA)4‐positive subclones could be isolated. Also, vector sequences were affected by alterations during the cloning process. These phenomena are discussed within the context of possible mechanisms for cloning artifacts.
Plant DNA fingerprinting with radioactive and digoxigenated oligonucleotide probes complementary to simple repetitive DNA sequences
Authors: Weising Kurt , Beyermann Birgit , Ramser Juliane , Kahl Günter
Published: May 2005

The existence of hypervariable DNA sequences in nuclear genomes, and the use of appropriate “fingerprinting” probes to detect them, has gained widespread scientific interest, and also led to multiple applications in diverse areas. Two years ago, the new technique of “DNA fingerprinting” was also introduced into the analysis and characterization of plant genomes, initially by using human or M 13 minisatellites as probes. In the present article, we demonstrate the applicability for plant DNA fingerprinting of oligonucleotide probes specific for simple repetitive DNA sequences. We show that various levels of intra‐ and interspecific polymorphisms can be detected; the information to be gained depends on the optimal combination of probe and species. Variety‐specific patterns were obtained in several cases. Some probes revealed variability between individuals. Somatic variability was not observed. Different DNA isolation and purification procedures were tested in order to introduce a fast and easy‐to‐perform isolation method suitable for a large variety of plant species. Nonradioactive fingerprinting was performed using digoxigenated oligonucleotides as probes. Banding patterns obtained with radioactive and digoxigenin‐based labeling techniques proved to be of similar quality.
On paternity determination from multilocus DNA profiles
Authors: Yassouridis Alexander , Epplen Jorg T.
Published: May 2005

Various approaches have been worked out to calculate to the probability of paternity from DNA multilocus profiles. Despite promising results, the application and practicability of these approaches are often restricted (due to, e.g., the equality assumption of the a priori probabilities by using Bayes theorem, no allowance of mutations, etc). Here we present two methods for paternity determination, one of which is an extension of the well‐known procedure of Evett et al. J. Forens. Sci. Soc. 1988, 28, 249–254. These methods depend on fewer assumption sand therefore maybe applied to a wide spectrum of disputed paternity cases.
Free mobility determination by electrophoresis in polyacrylamide containing agarose at a nonrestrictive concentration
Authors: Pospichal Jan , Vicchio Domenick , Chrambach Andreas
Published: May 2005

In the determination of the free mobility, related to the surface net charge, by quantitative gel electrophoresis, the previous arbitrary extrapolation of Ferguson plots from the lowest gel concentrations that give a mechanically stable gel to 0 %T has recently been replaced by measurement of mobilities across that concentration range, using the addition of 0.5 % agarose to polyacrylamide at the various low concentrations in application to a DNA fragment 155 bp in size (Orbán, L. et al., in preparation). The present study applies that approach to several proteins and DNA fragments smaller than 1300 bp, using 0.4 % agarose in polyacrylamide gels of varying concentration. The intercepts of the plots with the mobility axis provide experimental data by which the free mobility in polyacrylamide gel electrophoresis can be estimated for molecules not significantly retarded in their migration at the agarose concentration admixed to polyacrylamide. Across the gel concentration range below 3 %T, in the presence of agarose, the Ferguson plots of proteins and DNA fragments are convex. It was shown by mass spectrometry that this convex curvature of the plots in the mixed polymer is not significantly due to low polymerization efficiency in the concentration range of liquid polyacrylamide (below 3 %T).
Estimation of polyacrylamide gel pore size from Ferguson plots of normal and anomalously migrating DNA fragments. I. Gels containing 3 % N, N′‐methylenebisacrylamide
Authors: Holmes Diana L. , Stellwagen Nancy C.
Published: May 2005

The mobilities of normal and anomalously migrating DNA fragments were determined in polyacrylamide gels of different acrylamide concentrations, polymerized with 3 % N, N′‐methylenebisacrylamide as the crosslinker. The DNA samples were a commercially available 123‐bp ladder and two molecular weight ladders containing multiple copies of two 147‐base pair (bp) restriction fragments, obtained from the MspI digestion of plasmid pBR322. One of the 147 bp fragments is known to migrate anomalously slowly in polyacrylamide gels. Ferguson plots were constructed for all multimer ladders, using both absolute mobilities and relative mobilities with respect to the smallest DNA molecule in each data set. If the retardation coefficients were calculated from the relative mobilities, and the rms radius of gyration was used as the measure of DNA size, the Ogston equations were obeyed and the gel fiber parameters could be calculated. The effective pore sizes of the gels were estimated from the gel concentration at which the mobility of a given DNA molecule was reduced to one‐half its mobility at zero gel concentration. The estimated pore radii ranged from ∼ 130 nm for 3.5 % gels to ∼ 70 nm for 10.5 % gels. These values are much larger than the pore sizes previously determined for the polyacrylamide matrix.
Separation of human alloalbumain variants by isoelectric focusing
Authors: Rocha Jorge , Kömpf Jost , Ferrand Nuno , Amorim António , Ritter Horst
Published: May 2005

A technique for the separation of human alloalbumin variants by means of isoelectric focusing in the presence of 8M urea and 60 mM L‐serine is described. The potential usefulness of this technique in the detection and classification of genetic heterogeneity at the albumin locus is demonstrated by the differentiation of three human alloalbumin variants of European origin.