bluuebunny/crossref_metadata_2025_split huggingface dataset:
Isoelectric focusing of dansylated amino acids in immobilized pH gradients
Authors: Bianchi‐Bosisio Adriana , Righetti Pier Giorgio , Egen Ned B. , Bier Milan 
Published: May 2005

AbstractThe 21 free amino acids commonly encountered in proteins have been transformed into “carrier ampholyte” species by reacting their primary amino groups with dansyl chloride. These derivatives can thus be focused in an immobilized pH gradient covering the pH 3.1 to 4.1 interval, except for arginine, which still retains a pI of 8.8. Due to their inherent fluorescence, the dansyl derivatives are revealed in UV light, with a sensitivity of the order of 2–4 ng/mm2. All nearest neighbors are separated except for the following couples: Asn‐Gln, Gly‐Thr, Val‐Ile and Cys‐Cys2, with a resolving power, in a ΔpI scale, of the order of 0.0018 pH units. Except for a few cases (notably the aromatic amino acids) the order of pI values is well correlated with the pK values of carboxyl groups, suggesting that the latter are not altered by dansylation. From the set of pKCOOH ‐pI values of the different amino acids, the pK of the tertiary amino group in the dansyl label has been calculated to be 5.11 ± 0.06. Knowing the pK of the amino‐dansyl and the pI of the excess, free dansyl label (pI = 3.34) we have derived, for its sulfonic acid group, a pK of 1.57.
Microelectrophoresis in the differential diagnosis of proteinuric diseases
Authors: Weber Michael H. , Cheong (Kim) Kyung‐Sun , Schott Klaus‐Joachim , Neuholff Volker 
Published: May 2005

AbstractMicro‐polyacrylamide gel electrophoresis (micro‐PAGE) has been shown to be useful for molecular size separation of urinary proteins. In addition to the sodium dodecyl sulfate micro‐gradient gel electrophoresis in 10 μL capillaries, which is used routinely for examination of proteinuria in our laboratory, we tried micro‐gradient slab gel electrophoresis (micro‐slab‐PAGE) for molecular weight determination of urinary proteins. It was also employed for monitoring patients with proteinuric diseases. For detailed studied of urinary proteins, micro‐two‐dimensional‐electrophoresis is recommended.
High‐resolution two‐dimensional electrophoresis of myofibrillar proteins with immobilized pH gradients
Authors: Pernelle Jean‐Jacques , Chafey Philippe , Lognonne Jean‐Luc , Righetti Pier Giorgio , Bosisio Adriana Bianchi , Wahrmann Juan P. 
Published: May 2005

AbstractPrevious studies by chromatography and electrophoresis have demonstrated that myosin is composed of two classes of polypeptide chains: high relative molecular mass (Mr) components (200 000) and a class of lowerMrspecies, called the “light chains” (LC). It has been shown before by others that the slow‐twitching fibers contain 2 types of isoforms: LC1s and LC2s, while the fast‐twitching fibers contain 3 types: LC1f, LC3f and LC2f, the latter also existing as phosphorylated forms. In the present investigation, utilizing two‐dimensional (2‐D) maps generated with narrow immobilized pH gradients in the first dimension, a higher resolution of the myosin LC has been achieved. The LC1s chains are further fractionated into at least four isoforms; LC2s into three; LC2f into four and LC3f into three sub‐species in rabbit muscles. Skeletal muscle tropomyosin, which is separated into three forms (αs, αfand β), is here resolved into six components, while actin from skeletal muscle, previously described mainly as a single chain (α), is here sub‐fracionated into four isoforms of identicalMrand different charge. The series of isoforms here reported could represent a fine probe for investigating such complex phenomena as the cellular differentiation of human satellite cells from normal and pathological subjects in greater detail than before.
A novel highly sensitive electrophoretic method for the detection and quantification of proteinase activity
Authors: Poulsen Otto M. , Hau Jann 
Published: May 2005

AbstractA novel highly sensitive method for the detection and quantification of proteinase activity is presented. The proteinases were forced electrophoretically into the substrate (sodium caseinate) containing agarose gel. The formation of enzyme‐substrate complexes retarded the migration of the proteinases in the electric field and decreased the overall mobility of the enzymes. The proteinases were released as a result of the degradation of the substrate, and consequently the height of the obtained rocket shaped zones of hydrolysis were proportional to the concentration of proteinases in the samples. The electrophoretic method accelerated penetration of the substrate‐containing gel by the proteinases and subsequently resulted in a 100‐fold increase in sensitivity as compared to the radial diffusion method, depending entirely on diffusion.
The voltage across wide pH range immobilized pH gradient gels and its modulation through the addition of carrier ampholytes
Authors: Fawcett John S. , Chrambach Andreas 
Published: May 2005

AbstractIn pH 4–10 immobilized pH gradient (IPG) electrophoresis, one observes (a) partial failure of the sample to enter the gel; (b) which halt protein migration; (c) absence of defined transient state band patterns; (d) lateral zone spreading and distorted zone profiles; (d) uneven voltage profiles with a distinct voltage maximum; (f) difficulty in measuring pH profiles with the contact electrode. These problems are remedied through the addition of 0.1 to 1 % carrier ampholytes (CA) to the IPG gel. Problems (a) to (c) disappear in IPG tube gels. IPG in presence of CAs (ICAPG) of pI‐range 3.5–9.5 or 6–8 yields a pH gradient, a protein pattern and a pH gradient stability essentially the same as that in IPG gels of the same pH‐range 4–10. ICAPG is insensitive to ionic strength of the sample up to 0.1 M. Gel swelling and water exudation increase in ICAPG which make ICAPG in tube gels inapplicable when CA concentrations exceed 0.1 %.
Size, charge and structural heterogeneity of Brucella abortus lipopolysaccharides demonstrated by two‐dimensional gel electrophoresis
Authors: Sowa Blair A. , Crawforda Richard P. , Heck Fred C. , Williams John D. , Wu Albert M. , Kelly Katherine A. , Garry Adams L. 
Published: May 2005

AbstractPhenol extracted, alkali‐treated lipopolysaccharide (aLPS) from vaccine strain (S19) Brucella abortus was demonstrated by two‐dimensional gel electrophoresis to consist of at least ten silver staining, polydisperse analogues having different pIs. When tested on nitrocellulose immunoblots, all ten were antigenically reactive with bovine anti‐B. abortus polyclonal sera, but only six reacted with anti‐B. abortus O‐antigen murine monoclonal antibody. Analogues focusing at different pIs were concluded to arise from differences in either core or O‐antigen side chain structure or because of covalently bound protein. While not qualitatively different, aLPS from pathogenic B. abortus strain 2308 had lesser amounts of analogues 1, 2, 5, 6, and 8 than did aLPS from strain 19 (vaccine). The 2‐D gel electrophoresis method was demonstrated to be of value in the analysis of aLPS from B. abortus and may be useful in the study of lipopolysaccharides from other sources.
HERMeS: A second generation approach to the automatic analysis of two‐dimensional electrophoresis gels Part I: Data acquisition
Authors: Vincens Pierre , Paris Nicolas , Pujol Jean‐Luc , Gaboriaud Christine , Rabilloud Thierry , Pennetier Jean‐Louis , Matherat Philippe , Tarroux Philippe 
Published: May 2005

AbstractIn this series of papers, we describe the computer system we have developed for two‐dimensional (2‐D) gel analysis. Here the acquisition part of the system is described and a set of criteria able to measure the overall performance of the system is presented. These criteria are essentially based on frequency domain considerations using Fourier transforms. We show that the transfer function characteristics of the input system must be carefully checked to avoid any image distortion; we propose an easily computed criterion to obtain setting conditions giving a correct system response. These features are discussed considering the specificity of the 2‐D gel images that can be well modeled by a multi‐Gaussian surface. Then the visualization device and its main abilities to treat and modify the aspect of the images are presented. Statistical functions using second‐order image histograms are introduced. They can be used to look at the general aspect of the images, as a global and accurate criterion for quality checking. For this purpose, covariance, entropy and inertia have been found to be most suitable. Second‐order histograms give additional information on image anisotropy and can direct the user towards the convenient values of the parameters for the subsequent treatments. Coding is considered from both the point of view of image compression and image sending throughout a computer network. An efficient code protecting all the image informations is suggested and discussed.
Detection of soluble peroxidases by direct plant tissue isoelectric focusing
Authors: Vioque Blanca , López Antonio , Castellano José M. , Albi Miguel A. , Vioque Agustín 
Published: May 2005

AbstractRectangular sections of leaves, vacuum‐infiltrated with distilled water or aqueous solutions of potassium chloride, were applied directly onto the surface of polyacrylamide gels followed by isoelectric focusing. Soluble peroxidases (free and ionically bound) were released from the tissue on isoelectric focusing with resultant characteristic zymograms for the different plant tissues.
Reducing the background to silver‐stained polyacrylamide gels after two‐dimensional electrophoresis
Authors: Amess Robert , Peter Spragg S. 
Published: May 2005

AbstractA high background found after silver staining of polyacrylamide gels following two‐dimensional electrophoresis and generated from the cathode in the second‐dimensional separation has been investigated. Material is formed which is electrophoretically mobile and apparently derived from the buffer components. It behaves like a peptide as far as silver staining. The intensity of this background was reduced by treatment of the second‐dimensional gel with a photographic fixing agent prior to silver staining. Moreover, spots which previously appeared negatively stained were changed by the treatment and the result was a general improvement in the contrast.
Apolipoprotein E phenotyping from serum by Western blotting
Authors: Menzel Hans‐Jürgen , Utermann Gerd 
Published: May 2005

AbstractA new method for the apolipoprotein E (apo E) phenotyping directly from human serum has been developed. The method is based on isoelectric focusing of delipidated serum in vertical slab gel systems followed by blotting and immunostaining using polyclonal anti‐apo E antibodies as the first antibody. Apo E phenotyping with this method in 150 serum samples gave exactly the same results as obtained with the conventional method based on isoelectric focusing of delipidated very low density lipoproteins isolated by ultracentrifugation and detected by protein staining. Compared with the conventional method, the present method is less expensive, suitable for large‐scale screening purposes e. g. diagnosis of type III hyperlipidemia and needs only a few microliters of serum. By application for screening of hyperlipidemic sera, we were able to detect two patients with an apo E‐1 variant and two others with “new” E‐3 and E‐4 mutants by the described methods. In addition apparent molecular weight variants of apo E could be detected by sodium dodecyl sulfate‐gel electrophoresis of sera followed by blotting and immunostaining.
Combination of line immunoelectrophoresis and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis for molecular weight evaluation of isozymes: Application to iso‐α‐amylases identified in barley and rice seeds
Authors: Kerhardy Frédéric , Hara‐Nishimura Ikuko , Nishimura Mikio , Daussant Jean 
Published: May 2005

AbstractA procedure for enzyme purification by means of line immunoelectrophoresis compatible with subsequent sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of the purified enzyme and Coomassie Blue staining is described. Electrophoresis was carried out in an agarose gel backed to GelBond film. The enzyme activity was detected after immunoelectrophoresis on the wet gel and the immunoprecipitate by protein staining on the dried gel. The strip bearing the maximum amount of precipitate was excised and the dried agarose gel scratched off the GelBond support. The dried gel was extracted with the Laemmli sample buffer, heated at 100 °C for 10 min and submitted to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Proteins were stained with Coomassie Blue. Difficulties due to the presence of the immunoglobulin G subunits were encountered. The validity of the procedure was checked with an α‐amylase from barley purified by immunoaffinity chromatography. The procedure was used to compare the molecular weight of α‐amylase isozymes identified in barley and in rice seeds. In barley, α‐amylase I, the minor enzyme group appearing during germination, had a slightly higher molecular weight than α‐amylase II, the main enzyme group. In germinating rice seeds, the minor enzyme group, antigen D, was found to have a molecular weight slightly lower than the one of the other rice α‐amylases. A new α‐amylase antigen identified in rice as the constituent with the lowest pI was called pre‐A. Its molecular weight was found to be the same as the one of the main enzyme group, antigen (A + B).
HERMeS: A second generation approach to the automatic analysis of two‐dimensional electrophoresis gels. Part IV: Data base organization and management
Authors: Vincens Pierre , Tarroux Philippe 
Published: May 2005

AbstractA database structure is needed for easy handling of the large quantity of data obtained from a series of gel electrophoresis experiments and also for defining the first statements of a standard between experiments from different origins. This paper describes the database concept and the database interrogation strategies in the field of two‐dimensional electrophoresis and protein expression data. First, the main characteristics of our database structure and the main capabilities offered to the user to handle and interrogate the structure, as well as to retrieve his information are described. Gel standardization and protein identification are then examined. A relational database, associated with the description of each spot according to internal and external (physicochemical) descriptors, provides a suitable structure for a standardization procedure. A dynamic updating of the actual position of each spot, each time new positional information is entered in the system, allows the encoding of standardized positions. A database structure can also be used to establish a link between different kinds of information from different areas required for a correct interpretation of two‐dimensional gel data. Particularly, information from the gene sequence of a given protein may offer insights into the regulation of this protein. Thus a link between the two‐dimensional gel database and sequence databases is implemented in our system. The information available from sequence data may help to interpret electrophoresis data. Furthermore, a formal presentation of the database structure is achieved in order to propose a standardized description of the database structure and concepts used. This feature allows comparisons between different databases and frees the user from a specific configuration. Logical formulations are introduced using predicate calculus and then lead to a description of manipulation tools in terms of a declarative programming language.
HERMeS: A second generation approach to the automatic analysis of two‐dimensional electrophoresis gels. Part V: Data analysis
Authors: Tarroux Philippe , Vincens Pierre , Rabilloud Thierry 
Published: May 2005

AbstractData analysis programs are essential tools for processing the large quantity of data produced by two‐dimensional gel electrophoresis computerized analysis. Global handling of two‐dimensional gel information can be achieved using both analysis programs and artificial intelligence techniques. First, a statistical multivariate approach is presented, used to sort gels and to obtain a gel classification according to protein patterns. Both clustering and multivariate techniques are used and the results show that a classification of protein expression patterns according to effector action can be achieved. Spots can also be sorted and compared according to their expression under effector action. Each spot is thus described using a set of variables (its intensity for each gel pattern) and its description is essentially given in a multivariate way. Therefore, each facet of the regulation of a particular protein according to the action of several effectors can easily be taken into account. Spot sets containing spots having common regulation characteristics are obtained using principal component analysis. We show that each group corresponds to a functional aspect of the regulation in a given cell line. The map obtained describes the organization of cell functions and the interactions of these functions. Another approach employs artificial intelligence techniques. Since the rules available for describing some aspects of knowledge in the field of two‐dimensional electrophoresis remain unknown, learning techniques are suitable for defining them. Their use for the automatic recognition of spot groups belonging to the same entity on a gel is illustrated. Our learning program is applied to the determination of glycosylation groups and to the identification of relevant spot patterns.
Evaluation of recombinant human tumor necrosis factor by cell electrophoresis. I In vivo effect of recombinant human tumor necrosis factor on tumor cells, thymocytes and lymph node cells in tumor‐bearing mice
Authors: Knippel Eberhard , Rychly Joachim , Werner Heinz , Schütt Wolfgang , Klinkmann Horst 
Published: May 2005

AbstractTumor cells, thymocytes and lymph node cells in normal and tumor‐bearing mice treated with recombinant human tumor necrosis factor (rH‐TNF) were analyzed using automated cell electrophoresis to measure and classify cells according to cell surface charge. In the benzopyrene‐induced tumor system the intratumoral administration of rH‐TNF caused marked tumor necrosis and an increased cell surface charge on the tumor cells as detected by increased electrophoretic mobility (EPM). The thymus and lymph node are involved during murine tumor development characterized by abnormal weight and EPM pattern of lymphocyte histograms. Thymocyte and lymph node histograms were restored to their normal pattern by treatment of tumor bearers with rH‐TNF. The analysis of electrophoretic patterns of murine tumor cells, thymocytes and lymph node cells may be a simple method to evaluate drug effects on the immune system when automated techniques are applied.
A system for automated DNA electrophoresis, molecular hybridization and electronic detection: I Electrophoresis and hybridization
Authors: Zapolski Edward J. , Buas Michael , Golab Thomas , Ledley Robert S. , Gersten Douglas M. 
Published: May 2005

AbstractWe have designed and constructed a computer‐controlled, fully automated system for Southern‐type nucleic acid hybridization analysis. Restriction enzyme digests of DNA are placed into sample wells of the gel contained on a nine‐fingered plastic frame. The 32‐labeled probe is loaded into the hybridization chamber. Instructions for all the subsequent steps in the fully automated process are specified by the operator's answers to questions which appear on the computer screen at the start of the experiment. The system performs horizontal submarine electrophoresis. An adjustable endpoint detector concludes electrophoresis. Automatic voltage/temperature feedback control maintains maximum allowable voltage while keeping the temperature constant. Following electrophoresis a robot arm moves the gel frame from station to station. The system then affixes the separated fragments to a solid phase matrix, denatures, neutralizes, hybridizes, washes, dries and detects the 32 P according to the specifications preprogrammed by the operator. The results, printed out by the computer, give a plot of radioactivity versus distance from the origin for each of the nine simultaneous hybridizations.
Paraffin oil protected high resolution hybrid isoelectric focusing for the demonstration of substitutions of neutral amino acids in denatured proteins: The case of four human transthyretin (prealbumin) variants associated with familial amyloidotic polyneuropathy
Authors: Altland Klaus , Becher Pia , Banzhoff Angelika 
Published: May 2005

AbstractAll four sequenced variants of human plasma transthyretin (TTR) with different substitutions of electrically neutral amino acids and associated with familial amyloidotic polyneuropathy (i. e. TTR(Met30), TTR(Ala60), TTR(Tyr77) and TTR(Ser84)) have been separated from the normal TTR monomer by hybrid isoelectric focusing (HIEF) under denaturing conditions. The pI differences from the normal monomer were between 0.0025 and 0.005 pH units corresponding to 0.7–1.4 % of a full charge unit difference. The detectability of these variants by HIEF under denaturing conditions (7 M urea, 50 mM dithiothreitol, 2 % Triton X–100) was explained assuming interaction of the amino acids at the substitution site with a charged amino acid in its close vicinity. It is proposed to distinguish a high from a low level of resolution for HIEF under denaturing conditions to underline the fact that the studied variants could only be detected by extreme flattening of the pH gradient. To provide protection against modification by the laboratory atmosphere the entire gel, including the electrode wicks and sample application site, was covered with a layer of paraffin oil.
Thermal instability of electrophoresis gels
Authors: Woolley Paul 
Published: May 2005

AbstractUnder certain, defined conditions the temperature‐conductivity relationship of electrolytes leads to instabilities in the temperature distribution of electrophoresis gels if these are not efficiently thermostatted. These instabilities manifest themselves in practice in two ways: (i) as unequal temperatures of two identical gels connected in parallel and (ii) as an uneven temperature distribution across the face of individual gels. The latter is a principal cause of the undesirable “smile” effect that occasionally bedevils RNA and DNA sequencing gels. A quantitative description of these instabilities is put forward and suggestions for their avoidance are made.
Electrophoresis of ganglioside micelles
Authors: Catsimpoolas Nicholas , Griffith Ann L. 
Published: May 2005

AbstractGangliosides are known to exist as monomers in organic solvents and to aggregate in aqueous systems with the formation of high molecular weight micelles. It is shown here that the ganglioside micelles can be rendered visible by incorporation of a lipophilic dye and analyzed by conventional polyacrylamide gel electrophoresis. Mixed micelles of gangliosides and commonly used detergents as well as interaction products of ganglioside micelles with albumin were thus visualized by polyacrylamide gel electrophoresis for the first time.