bluuebunny/crossref_metadata_2025 huggingface dataset:
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

Data 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

Tumor 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

We 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

All 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

Under 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

Gangliosides 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.
Comparison of semi‐dry and conventional tank‐buffer electrotransfer of proteins from polyacrylamide gels to nitrocellulose membranes
Authors: Tovey Euan R. , Baldo Brian A.
Published: May 2005

The rate of transfer of radiolabelled proteins from sodium dodecyl sulphate‐polyacrylamide gels to nitrocellulose membranes by semi‐dry blotting and electro‐transfer in a tank of buffer with and without sodium, dodecyl sulphate was quantitated. The semi‐dry blot method transferred about two thirds of most proteins in the first hour; less than one tenth of the total protein in the gel was transferred in the following hour. Conventional transfer in a tank of buffer without sodium dodecyl sulphate was dependent on the molecular weight and isoelectric point of the proteins. The efficient migration of high molecular weight and basic proteins required the presence of sodium dodecyl sulphate. The efficiency of transfer to the nitrocellulose depended both on the transfer method and on the pore size of the nitrocellulose. Almost all protein which left the gel could be accounted for when 0.1 μm pore size was used, but when 0.45 μm pore size was used in combination with semi‐dry blotting or transfer in a tank of buffer containing 0.1 % sodium dodecyl sulphate, less than 60 % of the protein could be accounted for after transfer. When transfers by two of the methods were compared using a mite extract as an antigen source and a group of miteallergic sera for detection, the same proteins were detected in each case but those which had been transferred by the semi‐dry blot method were more easily detected by the antisera used.
The application of ligand overlay of protein blots to the study of the nicotinic acetylcholine receptor
Authors: Gershoni Jonathan M.
Published: May 2005

Protein blotting is used to identify and characterize the ligand binding site of the nicotinic acetylcholine receptor. The affinity of the complex formed on the filter can be determined by quantitative blot analyses. The precise binding‐sequence can be pinpointed by toxin overlay of proteolysed receptor α‐subunit. These techniques are now being employed in the analysis of bacterially expressed toxin binding sites.
Quantitative gel electrophoresis of polystyrene particles with 20–60 nm radii on 30% crosslinked polyacrylamide gel
Authors: Orbán László , Tietz Dietmar , Chrambach Andreas
Published: May 2005

The hypothesis was tested that polyacrylamide gel which is presumably of constant fiber structure within a practical range of gel concentrations would yield a linear Ferguson plot in the gel electrophoresis of particles in the size range of 10–100 nm. Polystyrene particles ranging from 22 to 60 nm in radius were subjected to gel electrophoresis on 30% N,N′‐methylenebisacrylamide‐crosslinked polyacrylamide (2.0 to 3.2% gel concentration) containing 50 mM 3‐[(3‐cholamidopropyl)‐dimethyl ammonio]‐1‐propane sulfonate (CHAPS). Visualization of the zones required transparent gels which were formed by polymerization both at 50 °C and in the presence of 6 M urea. Ferguson plots [log(mobility) vs. gel concentration] were progressively curved with increasing particle size from 22.4 to 59.4 nm radius. As in agarose gel electrophoresis, this curvature comprises one or several sigmoidal curve elements, presumably indicative of particle compression [1, 2]. However, by contrast with agarose gel electrophoresis, the curves can be aligned along a linear, rather than a convex axis, and may therefore be designated as “overall‐linear” in essential agreement with the hypothesis underlying this study. However, contrary to expectation, the polyacrylamide gel electrophoresis of particles in the size range of interest did not provide a practical method since the gel concentration range comprising Ferguson plots of the polystyrene particles in polyacrylamide is excessively narrow and the concentration range needed for extrapolation of the Ferguson plot to obtain free mobility values is excessively wide.
A convex “Ferguson plot” of polystyrene particles in electrophoresis on 0.25 to 2.0 % polyacrylamide (30 % Bis‐crosslinked)
Authors: Orbán László , Tietz Dietmar , Chrambach Andreas
Published: May 2005

The plot of log(mobility) vs. gel concentration for polystyrene particles of 26.9 and 44.2 nm radius in 30% Bis‐crosslinked polyacrylamide below 2.2 %T, containing 22.5% Metrizamide and 6 M urea, is convex. The convex plots of the two particles are essentially parallel. This parallelism suggests a retardation by a mechanism other than sieving. The parallel plots cover a range of polyacrylamide concentrations characterized by progressive fluidity as %T is lowered below 2 %T. The convex curvature renders suspect the previous free mobility and valence determinations in polyacrylamide gel electrophoresis obtained by linear extrapolation of Ferguson plots to 0 %T. Nonlinear extrapolation to 0 % T of plots of log(mobility) vs. gel concentration in the concentration range below 2 %T of 30% Bis‐crosslinked polyacrylamide for the two polystyrene particles, corrected for the effects on mobilities of Metrizamide, urea and a lower ionic strength, provides free mobility values similar to those obtained under otherwise identical conditions on agarose gel electrophoresis.
Affinity chromatography of reticulocyte lysates on haptoglobin improves two‐dimensional mapping of translation products
Authors: Rademacher Bruce E. , Steele William J.
Published: May 2005

A simple method is described for enhancing the resolution and detectability of translation products in two‐dimensional mapping of reticulocyte lysate translation systems. Assays are fractionated by affinity chromatography on human haptoglobin‐Affi‐Gel 15 to remove hemoglobin and translation products are eluted with sodium dodecyl sulfate, buffered at pH 9.2. Translation products purified in this, way have less than 10 % of the hemoglobin present in the original assay. The recovery of translation products is about 95 % after haptoglobin chromatography and about 75 % after concentration for mapping. Hemoglobin removal not only enhances the resolution and detectability of low and moderate abundance translation products but also reduces horizontal and vertical streaking in two‐dimensional mapping without significantly altering the distribution of translation products.
A2HS*11: A new allele of alpha‐2‐HS‐glycoprotein found in Afro‐Caribbeans
Authors: Westwood Sara A. , Seaman Philip J. , Ablett Peter J. , Yuasa Isao , Weidinger Sebastian , Umetsu Kazuo
Published: May 2005

A novel and unique protein pattern for alpha‐2‐HS‐glycoprotein (A2HS) was found in the sera from two unrelated Afro‐Caribbean donors after isoelectric focusing followed by immunofixation. In the native state the variant bands were located between A2HS 7 and A2HS 10 bands. The desialized samples showed that a cathodal variant band was indistinguishable from the A2HS 5 band. These findings indicated that both these donors possessed a variant phenotype of A2HS determined by a new allele A2HS*11, in combination with the common A2HS*1.
Analysis of two‐dimensional electrophoretic protein patterns using a video camera and a computer. II. Adaptation of automatic spot detection to visual evaluation
Authors: Prehm Joachim , Jungblut Peter , Klose Joachim
Published: May 2005

Three two‐dimensional electrophoretic protein patterns (fluorograms) showing a representative spectrum of different qualities of resolution (spots well separated, spots embedded in “streaks” and spots of low contrast to the background) were selected to ascertain the optimum of the parameters of a computer program used for spot detection with a video camera/computer system. A “visual norm‐pattern” was constructed on the basis of the results obtained by visual evaluation of the three patterns by several test persons. The visual norm‐pattern (a graphic spot pattern) consisted of two classes of spots: “visible spots” (spots detected by all test persons) and “invisible but true spots” (spots detected by all persons, but only if the exposure time of the X‐ray film was increased). This pattern was used as a standard pattern for automatic evaluation. The automatic evaluation of the three test patterns was performed under various conditions: In the computer program two parameters critical for spot detection, parameter P1 (responsible for the separation of the spots) and P2 (responsible for the size of the spots), were varied over a broad range. In this way a third class of spots, “invisible, artifactual spots”, was revealed. The three spot classes could not be distinguished by means of the automatic system. Therefore, the visual norm‐pattern was used to adjust parameters P1 and P2 at an optimum level of sensitivity. The optimum values found for P1 and P2 allowed us to detect all the visible spots of a two‐dimensional protein pattern, and, in addition, a portion of the invisible but true spots, while none of the artifactual spots was included in the computer‐produced patterns. The P‐values chosen could be used for two‐dimensional protein patterns of different qualities. Slight deviations of the P‐values from the optimum were acceptable.
Rat liver cytosolic protein changes after ethanol exposure studied by two‐dimensional electrophoresis
Authors: Wirth Peter J. , Vesterberg Olof
Published: May 2005

Rats were fed liquid food containing ethanol in concentrations ranging from 1–5 % for 13 weeks. Livers were removed for histopathology and the liver cytosolic protein fraction was prepared and used for two‐dimensional gel electrophoresis (2D‐PAGE). Polypeptides were visualized by silver staining. Scanning was made for estimation of the relative abundance of protein in each polypeptide spot in the gels and for comparison between rats. Visual inspection and scanning of gels with the stained polypeptide spots obtained after equilibrium isoelectric focusing and non‐equilibrium pH gradient electrophoresis revealed that: (1) within the control rat and ethanol‐treated rat livers the numbers of polypeptide spots detected using isoelectric focusing in the first dimension were approximately 500 and for non‐equilibrium pH gradient electrophoresis 400; (2) in the control group the variation in the estimated amount of protein in each spot was remarkably small; (3) pronounced differences in the relative abundance of protein in several of the spots was observed in the ethanol‐exposed rats as compared to controls. Dose‐response relations and possible causes for the effects of ethanol are discussed.
Different patterns of human serum transferrin on isoelectric focusing using synthetic carrier ampholytes or immobilized pH gradients
Authors: D'Alessandro Anna M. , D'Andrea Gabriele , Oratore Arduino
Published: May 2005

Isoelectric focusing (IEF) of human serum transferrin allows splitting of the protein pattern into three forms corresponding to the diferric, monoferric and apoform. A detailed analysis of this pattern, performed on transferrin at different degrees of iron saturation, demonstrated that free Ampholine carrier ampholytes (CA) alter the expected results, always giving a complex pattern with multiple bands. In particular, the monoferric form appears to be the predominant one, regardless of the starting saturation of transferrin. In contrast to IEF‐CA, the new technique of IEF in immobilized pH gradients (IPG), shows a much simpler pattern with the same samples. Moreover, the different transferrin forms are focused at the same pI values as in IEF‐CA but the pattern appears to correspond to the expected distribution. IPG analysis gives a pattern similar to IEF‐CA when free Ampholine CA are added either to the samples and/or as electrode solutions. A chelating action of Ampholine CA on Fe+3 might be responsible for these effects, while Immobilines, due to their different chemical nature or to the different focusing procedure, are not able to interact with iron.
Production of monospecific antisera by immunization with precipitin line leads to misinterpretation of immunoblot
Authors: Aoues Abdelkader , Huault Claude
Published: May 2005

Immunoblotting displays an artifact in the preparation of monospecific antibodies from immunocomplexes cut out of line immunoelectrophoresis. Agarose induces the formation of antibodies towards a 68 K Da protein which is contained in commercial agarose.
Calculating “threeborate” ion mobilities at different ionic strengths and temperatures
Authors: Michov Budin M.
Published: May 2005

The mobilities of the “threeborate” ion at different temperatures and ionic strengths are calculated using previously published equations for calculating the mobilities of composed ions.
Comparison of oligoclonal immunoglobulin G bands in multiple sclerosis cerebrospinal fluid by immunoblotting and immunofixation
Authors: Mehta Pankaj D. , Patrick Bruce A. , Black Jeff
Published: May 2005

We previously analyzed unconcentrated cerebrospinal fluid (CSF) from patients with multiple sclerosis (MS) and other neurologic diseases by isoelectric focusing in agarose gel. We have now developed an immunoblot method for detection of oligoclonal IgG bands in unconcentrated MS CSF. The oligoclonal IgG band patterns seen after immunoblotting were compared with those of conventional immunofixation. Although immunoblotting was found to be rapid the resolution and intensity of oligoclonal IgG bands were somewhat better after immunofixation. Since immunofixation is simpler than immunoblotting, we recommend that clinical laboratories use immunofixation after isoelectric focusing to detect oligoclonal IgG bands in CSF.
Discontinuous buffer systems optimized for the agarose gel electrophoresis of subcellular particles
Authors: Orbán László , Hahn Elisabeth , Chrambach Andreas
Published: May 2005

Discontinuous buffer systems operative between pH 5.7 and 7.4, 0 °C, were generated, which are characterized by more rapidly displaced moving boundaries than applied previously. These allow one to resolve subcellular particles relatively rapidly and at relatively low agarose gel concentrations. A commercial mixture of DNA restriction fragments pre‐stained with ethidium bromide was found to be a suitable tracking dye for these boundaries.
Thin‐Layer agarose isoelectric focusing of alkaline phosphatase isoenzymes from human neutrophils
Authors: Brisson‐Lougarre Andrée , Vergnes Henri , Grozdea Jean , Biermé Robert
Published: May 2005

An improved method for thin‐layer agarose isoelectric focusing of alkaline phosphatases (AP) from human neutrophils is described. The solubilization of AP isoenzymes was studied with four detergents. The best results were obtained after sonication with Zwittergent 3–12 (1 % final concentration), followed by butanol extraction and ultracentrifugation 105 000 × g for 1 h. The cytosol can be stored at O °C or −80 °C, but not at −20 °C. Dialysis of the cytosol against a Tris buffer, pH 7.5, was imperative prior to focusing for removal of the detergent. Enzyme visualization is enhanced by incorporation of Zn Cl2 (3 mM) into the agarose gel. The focusing patterns consist of two sets of isoenzymes: two main zones with pI 6.4 and 6.8 and minor components with pI 4.2, 4.8 and 5.2.
A comparative study of protein synthesis by keratinocytes and fibroblasts in vitro using two‐dimensional gel electrophoresis and dual isotope autoradiography
Authors: Easty David J. , Patel Ketan , Dover Robin , Evans David J. , Dunn Michael J.
Published: May 2005

A procedure is described for the detection of keratinocyte specific proteins. Fibroblasts and keratinocytes were isolated from human skin and radiolabelled in vitro. Samples were separated by two‐dimensional polyacrylamide gel electrophoresis to compare the proteins synthesised by the different types of cultured skin cells. Dual label autoradiography of samples radiolabelled with [35S]methionine and [75Se]selenomethionine was used to identify keratinocyte specific proteins. We report 45 keratinocyte‐specific components and identify some of these proteins. The differential expression of these proteins and their relevance to epidermal differentiation are discussed.
Conditions for improved adsorption of calmodulin to nitrocellulose: Detection by 45Ca binding
Authors: Hincke Maxwell T.
Published: May 2005

The 45Ca filtration method which allows calcium‐binding proteins to be detected after their adsorption to nitrocellulose filters was used to test conditions for improved Western blotting of calmodulin. Our results indicate that dodecyl sulphate is not efficiently removed from calmodulin during electroelution in standard transfer buffers. This detergent disrupts binding of calmodulin to nitrocellulose. Electrotransfer in potassium buffer which precipitates dodecyl sulphate leads to a 6‐fold increase in calmoculin retention by nitrocellulose. With this and other modifications, the detection limit of the 45Ca overlay method of Maruyama et al. (J. Biochem. 1984, 95, 511–519) for calmodulin is increased to 0.2 μg per lane.
Genetic variations of human serum β2‐glycoprotein I demonstrated by isoelectric focusing
Authors: Richter Antoinette , Cleve Hartwig
Published: May 2005

Human serum β2‐glycoprotein I was analyzed by isoelectric focusing followed by im‐munoprinting or immunoblotting with monospecific antiserum. Isoelectric focusing revealed a heterogeneous pattern consisting of 4 major and 4–5 minor bands with isoelectric points of the major bands between pH 5.4 and 6.2. Comparative analysis of sera from more than 400 healthy blood donors showed individual variations of band patterns: six different phenotypes were observed. A family study of 44 families with a total of 129 children demonstrated the genetic control of this variation. Presumably, three alleles, called B2G*1, B2G*2, and B2G*3, determine six phenotypes: B2G 1, 2, 3, 1–2, 1–3 and 2–3. The phenotype 3, however, has not been found in this study. An additional phenotype, noted in one serum specimen, was tentatively classified as B2G 2–4. The distribution of phenotypes and alleles in two populations, from Munich and from Tyrol, has been examined and the frequencies are presented. This genetic polymorphism appears not to be associated with inherited quantitative variations of β2‐glycoprotein I found earlier. The inherited variations can still be recognized after treatment of sera with neuraminidase and with endoglycosidase F, although the banding pattern is altered and shifted towards the cathode. The genetic polymorphism can, therefore, not be ascribed to variations residing in the carbohydrate side chains.
Subtyping of group specific component (GC) in microbloodstains and semen stains by isoelectric focusing in ultrathin immobilized pH gradient gels followed by enzyme immunodetection
Authors: Pflug Werner
Published: May 2005

A powerful method for group specific component (GC) subtyping with good resolution of GC bands by isoelectric focusing on ultrathin immobilized pH gradients is described. After separation, GC detection is achieved using a highly sensitive alkaline phosphatase conjugated enzyme‐immuno system. The efficiency of the method in forensic case work for subtyping GC from diluted bloodstain extracts, blood microstains and semen stains is demonstrated. Furthermore, GC subtyping and selective detection of human GC provides the evidence for human origin of the stain. This is important when analyzing microstains with limited stain consumption.
The adsorption of large proteins in electrofocusing on immobilized pH gradients: I. Protein specificity and dependence on Immobiline and carrier ampholyte concentrations
Authors: Fawcett John S. , Chrambach Andreas
Published: May 2005

Phycoerythrin, ferritin, urease, β‐galactosidase and thyroglobulin, with molecular masses in excess of 200 kDa, adsorb and consequently fail to migrate to, and focus at, their pI positions in electrofocusing in immobilized pH gradients at a total Immobiline concentration of 20 mM while they do focus normally in pH gradients formed by carrier ampholytes. The addition of carrier ampholytes (pH range 3.5–9.5) at concentrations of 0.1 to 5 % to the Immobiline‐containing gels reduces adsorption (desorbs) some but not all of the 5 proteins at specific Immobiline concentrations. The adsorption is not due to water redistribution and consequent reduction in gel porosity; nor is it due to conductivity minima across the pH gradient. The hypothesis that the presence of oligomeric Immobiline contributed to the protein adsorption is the subject of the accompanying report.
Fast and sensitive protein staining with colloidal acid Violet 17 following isoelectric focusing in carrier ampholyte generated and immobilized pH gradients
Authors: Patestos Nikos P. , Fauth Markus , Radola Bertold J.
Published: May 2005

A new method is described for fast and sensitive staining of proteins following isoelectric focusing in carrier ampholyte and immobilized pH gradient polyacrylamide gels. After fixation with trichloroacetic acid the gels are stained for 5–10 mm with 0.1–0.2 % colloidal Serva Violet 17 (generic name: Acid Violet 17; Color Index No. 42 650) in 10 % w/v phosphoric acid. After staining for only 0.5–3 min, major zones, corresponding to 100–500 ng protein, are visible without destaining on a weak background. Detection of minor components requires destaining with 3 % w/v phosphoric acid for 5–80 min depending on gel thickness (120–500 μm) and type of support (fabric reinforcedversusgels backed to a polyester film). For selected pH marker proteins (bovine serum albumin, carbonic anhydrase, horse myoglobin) a staining sensitivity of 1–2 ng/mm2protein is found. Dye elution from stained fabric reinforced gels with 50 % v/v dioxane‐water, followed by absorbance measurements results in a linear relationship over a range of 1–100 μg marker proteins. Staining colloidal Serva Violet 17 is the only method available for fast and high sensitivity and low background staining of immobilized pH gradient gels, without interference from selective dye binding in different pH ranges. Staining with the colloidal dye is convenient by avoiding organic solvents with unpleasant vapors and potentially hazadous.
Two‐dimensional electrophoresis. Analysis of tubulin proteins and peptides in neuronal and non‐neuronal tissues using immobilized pH gradients
Authors: Field Deborah J. , Lee James C.
Published: May 2005

The accurate identification of the individual protein products of multi‐gene families is essential to the interpretation of data from a wide range of experimental approaches including molecular biology, protein chemistry, and cell biology. We have adapted immobilized pH gradient isoelectric focusing to provide high resolution of tubulin proteins [1, 2]. Here we use these techniques to investigate the heterogeneity of tubulin in several neuronal and non‐neuronal tissues to provide an accurate evaluation of isotubulin composition. Of the ten sources examined, the greatest number of isotubulins was found in whole adult brain. Tubulin isolated from either neonate human or rat brain consists predominantly of the more basic alpha and beta isotubulins found in adult brain. Cerebrum, cerebellum, medulla and caudate nucleus all contain the same large number of isotubulins as in whole brain, but in varying proportions. Liver, kidney and spleen isotubulin populations are all similar to each other and consist of a simpler distribution than any neuronal tissue examined. The majority of the tubulin protein in these non‐neuronal tissues is composed of only the most basic alpha tubulins and intermediately‐charged beta tubulins. No isotubulins were identified that were unique to these three non‐neuronal tissues. Tubulin from neuroblastoma cells has an isotubulin distribution grossly similar to non‐neuronal sources but additionally contains two basic beta isotubulins found in adult brain that are absent from non‐neuronal tissues.
Isoelectric focusing of apolipoproteins in immobilized pH gradients: Improved determination of apolipoprotein Ephenotypes
Authors: Baumstrak Manfred W. , Berg Aloys , Halle Martin , Keul Joseph
Published: May 2005

A method for isoelectric focusing of apolipoprotein Ein an immobilized pH gradient with added carrier ampholytes has been developed. This method is an improvement over conventional isoelectric focusing of apolipoprotein E with respect to resolution reproducibility, and simplicity. Since monosialo isoforms are resolved from the not mally cofocusing asialo isoforms, unique patterns are obtained for all 6 common apolipoprotein E phenotypes. The method can also be applied to the screening of apolipoprotein A and C isoforms. Delipidated very low density lipoproteins (VLDL) have been used as the source of apolipoprotein E and C. Apolipoprotein A isoforms were focused directly from detergent‐treated serum. Immunodetection of apolipo protein E using capillary transfer was found to be compatible with the described method.
Isoelectric focusing in immobilized pH gradients with carrier ampholytes added for high‐resolution phenotyping of bovine β‐lactoglobulins: Characterization of a new genetic variant
Authors: Krause Ingolf , Buchberger Johann , Weiß Günther , Pflügler Monika , Klostermeyer Henning
Published: May 2005

The genetic variants of bovine β‐lactoglobulin (β‐lg) from the “Murnau‐Werdenfelser” breed were analyzed in three different isoelectric focusing (IEF) systems. While carrier ampholyte IEF with a pH gradient of 0.2 pH/cm did not resolve the new variant W from the B variant and IEF in immobilized pH gradients (IPG) with 0.1 pH/cm only partially resolved it, adequate separation was achieved with IPG‐IEF in a pH 5.25‐pH 5.7 gradient, in presence of 0.8 % w/v carrier ampholytes, both over a 10 and 17 cm separation distance. Apparent isoelectric points (pI's) and genetic frequencies (f) were as follows: β‐lg A, pI = 5.370,f = 0.364; β‐lg B, pI = 5.485,f = 0.480; β‐lg W, pI = 5.492,f = 0.076; and β‐lg D, pI = 5.610,f = 0.080. The small difference of Δ pI = 0.007 between β‐lg B and β‐lg W respectively, seems to originate from a “silent” substitution of neutral amino acid residues as compared to the larger Δ pI's of the other genetic variants of β‐lg, which result from substitution of charged amino acids.
Isoelectric focusing of alpha‐1 acid glycoprotein (orosomucoid) in immobilized pH‐gradients with 8 M urea: Detection of its desialylated variants using an alkaline phosphatase‐linked secondary antibody system
Authors: Eap Chin B. , Baumann Pierre
Published: May 2005

A method allowing a clear separation of the different variants of desialylated alpha 1‐acid glycoprotein (orosomucoid) has been developed using isoelectric focusing in immobilized pH gradients, supplemented with 8 M urea and 2 % v/v 2‐mercaptoethanol. Immunoblotting with two antibody‐steps afforded high sensitivity and permitted the detection of about 700 pg of alpha 1‐acid glycoprotein in a 20 μL plasma sample diluted 1:28 672. A one year old bloodstain, kept at room temperature, could easily be phenotyped.
Photocontrol of organelle and cell type specific changes in the polypeptide composition of Euglena and sorghum
Authors: Monroy Antonio F. , Gardiner William E. , Schwartzbach Steven D.
Published: May 2005

Two‐dimensional (2‐D) gel electrophoresis has been used to follow changes in cell type specific and organelle localized polypeptides upon exposure of etiolated sorghum shoots and dark‐grown resting Euglena to light. Total protein extracted from isolated bundle sheath strands and mesophyll protoplasts was resolved by 2‐D gel electrophoresis. The cell type specific polypeptides were localized on the whole shoot 2‐D gel map in order to determine changes in the levels of these polypeptides upon light exposure. An image analyzer was used to analyze fluorographs of 2‐D gels of total Euglena protein pulse‐labeled with [35S]sulfate in the dark, immediately upon light exposure and 1, 4, 14, 24, 48 and 72 h after light exposure. The subset of polypeptides whose relative rats of synthesis changes more than threefold immediately upon light exposure was i dentified. The different patterns of changes in the rate of synthesis of this subset of polypeptides were followed.
Two‐dimensional gel analysis of yeast proteins: Application to the study of changes in the levels of major polypeptides of Saccharomyces cerevisiae depending on the fermentable or nonfermentable nature of the carbon source
Authors: Bataillé Nelly , Thoraval Didier , Boucherie Hélian
Published: May 2005

Taking advantage of the recent identification of polypeptides of the carbor meta. bolism machinery on the yeast protein map [1], we applied two‐dimensional gel electrophoresis to a study of changes in protein composition of Saccharomyces cerevisiae depending on the fermentable or nonfermentable nature of the carbon source. The levels of the 250 most abundant polypeptides were compared. Thirty‐three were found to display markedly increased levels during growth on nonfermenable carbon sources. These 33 polypeptides include 11 mitochondrial polypeptides and polypeptides corresponding to alcohol dehydrogenase II, acetyl‐CoA synthetase, phosphoenol pyruvate kinase and hexokinase PI. Sixteen other polypeptides, in contrast, reached their higher levels during growth on fermentable corbon sources. Among these were identified the monomeric subunits of 6 giycoytic enzymes. Collectively the 33 polypeptides of the first class comprised over 30% of the total soluble proteins of cells grown on nonfermentable carbon source and 3 % during growth on fermentable carbon source. The protein fraction of the 16 polypeptides of the second ass corresponded to 10 % and 38 %, respectively. Together these results show that two‐dimensional gel electrophoresis, when coupled with the identification of polypeptides of the carbon metabolism apparatus, provides a valuable tool for approaching questions concerning carbon metabolism in S. cerevisiae.
Polypeptide changes induced by salt stress, water deficit, and osmotic stress in barley roots: A comparison using two‐dimensional gel electrophoresis
Authors: Hurkman William J. , Tanaka Charlene K.
Published: May 2005

Two‐dimensional polyacrylamide gel electrophoresis was used to analyze and compare the effects of short term treatments (24 h) of salt stress, water deficit (desiecation), and osmotic stress (polyethylene glycol and mannitol) on protein synthesis in roots of barley seedlings (Hordeum vulgare L. cv. CM 72). These comparisons were made to determine if the polypeptides of Mr 26 000 and 27 000 and pI of 6.3 and 6.5 that were observed previously to increase significantly with salt stress (Plant Physiol. 1987, 83 517–524) also increased with water deficit and osmotic stress. The polypeptide patterns for control‐and stress‐treated plants were qualitatively similar, but the net synthesis of a number of polypeptides was quantitatively altered by each of the stress treatments. Of the polypeptide changes induced by the stress treatments, many were unique to a specific stress. Other polypeptide changes were common between two or more of the stress treatments. Only one polypeptide change, a decrease, was common to all of the stress treatments. An important finding was that polypeptides that increased significantly in response to salt stress did not increase in response to water deficit or osmotic stress.
The use of a bivalent counter anion to control the effective mobility of the hydrogen ion constituent in cationic isotachophoresis
Authors: Sudor Ján , Stránský Zdeněk , Chmela Zdeněk , Boček Petr
Published: May 2005

The use of a bivalent counter anion for cationic isotachophoresis was studied both theoretically and experimentally. A mathematical model was proposed and solved to give the effective mobility of the hydrogen ion constituent, uH.H, in the respective electrolyte systems. The theoretical values agreed well with the experimental data in a series of electrolyte systems. The dependence of uH.H on such parameters as pH of the leading electrolyte, ionic mobilities of leading and counter ions, and dissociation constants was calculated and discussed. A bivalent counter anion will prove useful for the separation of low mobility cations and weak bases, as shown for the separation of substituted anilines in a succinate electrolyte system.
GESA – a two‐dimensional processing system using knowledge base techniques
Authors: Rowlands David G. , Flook Alan , Payne Peter I. , van Hoff Arthur , Niblett Tim , McKee Sean
Published: May 2005

The successful analysis of two‐dimensional (2‐D) polyacrylamide electrophoresis gels demands considerable experience and understanding of the protein system under investigation as well as knowledge of the separation technique itself. The present work concerns the development of a computer system for analysing 2‐D electrophoretic separations which incorporates concepts derived from artificial intelligence research such that non‐experts can use the technique as a diagnostic or identification tool. Automatic analysis of 2‐D gel separations has proved to be extremely difficult using statistical methods. Non‐reproducibility of gel separations is also difficult to overcome using automatic systems. However, the human eye is extremely good at recognising patterns in images, and human intervention in semi‐automatic computer systems can reduce the computational complexities of fully automatic systems. Moreover, the expertise and understanding of an “expert” is invaluable in reducing system complexity if it can be encapsulated satisfactorily in an expert system. The combination of user‐intervention in the computer system together with the encapsulation of expert knowledge characterises the present system. The domain within which the system has been developed is that of wheat grain storage proteins (gliadins) which exhibit polymorphism to such an extent that cultivars can be uniquely identified by their gliadin patterns. The system can be adapted to other domains where a range of polymorpic protein sub‐units exist. In its generalised form, the system can also be used for comparing more complex 2‐D gel electrophoretic separations.
The two radii of a charged particle
Authors: Michov Budin M.
Published: May 2005

The existence of two radii of each charged particle – a geometric and electrokinetic radii, is supposed. The mathematical relationship between them in the four possible combinations of an ion and its counterion is analyzed: (i) at equal geometric radii and, in absolute values, equal valencies; (ii) at equal geometric radii and, in absolute values, different valencies; (iii) at different geometric radii and, in absolute values, equal valencies; (iv) at different geometric radii and, in absolute values, different valencies. One of the equations worked out can be used to define the relationship between the geometric and electrokinetic radii of a polyion. All the equations are used in working out precise calculations.
Fast and high resolution analysis of human serum transferrin by high performance isoelectric focusing in capillaries
Authors: Kilár Ferenc , Hjertén Stellan
Published: May 2005

Human serum transferrin is a mixture of isoforms (isoproteins) having different amounts of carbohydrates. Each isoform may exist in iron‐free and iron‐complexed molecular form. The genetic variations in different populations increase the number of combinations of the different forms of transferrin. To resolve the many components in transferrin preparations, the new high performance capillary technique was employed for isoelectric focusing. Iron‐free transferrin and transferrin samples of known iron content were examined. The above method gives an exceptionally rapid analysis (within 15–25 min) of small amounts of samples (less than 1 μg protein) and as good as or better resolution than other isoelectric focusing techniques previously used for transferrin analysis. By monitoring the focused protein zones at both 280 and 460 nm the molecular forms of transferrin (iron‐free, monoferric and differic complexes) can easily be identified. Both steps of isoelectric focusing in capillaries (i. e., prefocusing and mobilization) can be used for analysis. We observed that chelating agents (e. g., carrier ampholytes, nitrilotriacetate) may release iron from micro‐syringes having metal pistons causing the formation of iron‐transferrin complexes.
Improved separation of alpha chains of collagen type I, type III, and type V by noninterrupted electrophoresis using thioglycolic acid as a negatively charged reducer
Authors: Nakamura Kazuyuki , Inoue Shin‐Ichi , Abiko Seishou , Aoki Hideo , Takeo Kazusuke
Published: May 2005

Improved separation of alpha chains of collagen type I (α1[I]2 α2[I]), type III(α1[III]3), and type V(α1[V] α2[V]α3[V]) was achieved by noninterrupted sodium dodecyl sulfate‐polyacrylamide gel electrophoresis with a negatively charged reducer, thioglycolic acid. The thioglycolic acid, added to the running buffer of the cathodic reservoir, in the middle of electrophoresis quickly migrated in the gel anode, reducing interchain disulfide linkages in collagen type III and dissociating it into its alpha chain monomer, α1[III], without an interruption of electrophoresis. The alpha chain, α1[III], migrated more slowly than the α1[I] and α2[I] chains of collagen type I, resulting in an excellent separation of α1[III] from α1[I]. The mobility of α1[III] could be controlled by varying the time of thioglycolic acid addition to the running buffer. This enabled us not only to separate α1[III] from α1 [I] and α1[V], but also to precisely quantitate these alpha chains, even at low protein loading of mixed samples.
Enzyme activity electrophoresis and rocket immunoelectrophoresis for the qualitative and quantitative analysis of Geotrichum candidum lipase activity
Authors: Jacobsen Tomas , Poulsen Otto M. , Hau Jann
Published: May 2005

The development and application of a rocket immunoelectrophoretic and an enzyme activity electrophoretic assay for the quantitative and qualitative analysis of Geotrichum candidum lipase activity is presented. The sensitivities of the four assays were (in arbitrary units): enzyme activity electrophoresis, 1–0.5; rocket immunoelectrophoresis, 0.5–0.2; radial diffusion, 1; titrimetry, 1. The electrophoretic methods made it possible to distinguish between high and low molecular weight forms of the G. candidum lipases. The enzyme activity electrophoretic methods can be combined with other electrophoretic techniques, as demonstrated here with isoelectric focusing, and produce useful information on physico‐chemical differences between different molecular forms of the lipase, e. g. forms with different pI.
Effect of electric field switching on the electrophoretic mobility of single‐stranded DNA molecules in polyacrylamide gels
Authors: Lai Eric , Davi Noelle A. , Hood Leroy E.
Published: May 2005

We have examined the effects of pulsed electric fields on the separation of single‐stranded DNA molecules in polyacrylamide sequencing gels. Using different electric field pulsing regimens, the mobilities of single‐stranded DNA molecules can be retarded or increased as compared to conventional electrophoresis. These results indicated that pulsed field techniques can be applied to gel electrophoresis of small single‐stranded DNA molecules.
Protein database development: Identification of glycosylated and phosphorylated proteins in unfractionated rat fibroblast lysates
Authors: Krauss Marc R. , Collins Paul J.
Published: May 2005

In this paper, techniques are demonstrated for unambiguously mapping phosphoproteins and glycoproteins into a protein database of unfractionated fibroblast proteins. These methods allow for precise registration of subsets of proteins with the image of total proteins. The methods are also applicable to database mapping of antigens identified by indirect immunodetection methods and receptor molecules identified with photoaffinity ligands.
A simple method for accurately locating and cutting strips from the isoelectric focusing gel during two‐dimensional electrophoresis using the phastsystem
Authors: Schiff Theodore A. , Freedberg Irwin M.
Published: May 2005

A rapid and simple method has been developed for precisely locating and cutting 1–3 mm strips from the isoelectric focusing gel during two‐dimensional electrophoresis using the PhastSystem. The protocol utilizes an easily constructed styrofoam template with marks to guide accurate cutting of the strips from the sample lanes. This system greatly enhances the reproducibility and quality of the two‐dimensional patterns obtained.