bluuebunny/crossref_metadata_2025_split huggingface dataset:
Tissue‐specific variation of pea mitochondrial polypeptides detected by computerized image analysis of two‐dimensional electrophoresis gels
Authors: Humphery‐Smith Ian , des Francs‐Small Catherine Colas , Ambart‐Bretteville Françoise , Remy René 
Published: May 2005

AbstractA tissue‐specific variation of a total of 152 mitochondria‐associated polypeptides is detailed for etiolated leaves, epicotyls and roots from the pea, Pisum satisum L., using computerized image analysis. These organ systems possess, respectively, 128, 100, and 96 mitochondria‐associated polypeptides with 38, 8, and 10, respectively, being unique to each. Seventy‐one polypeptides were observed to be present in all gels, but some of these also underwent a variety of quantifiable changes. Two polypeptides were identified that varied similarly to a polypeptide of interest (W), known to be abundant in pea epicotyls and potato tubers. It is postulated that these latter three polypeptides are therefore probably related functionally.
Resolution of circular, nicked circular and linear DNA, 4.4 kb in length, by electrophoresis in polyacrylamide solutions
Authors: Garner Mark M. , Chrambach Andreas 
Published: May 2005

AbstractCircular DNA of more than 1,400 bp in size is known not to migrate into polyacrylamide gels. The migration of supercoiled plasmid pBR322 DNA (4,363 bp) into uncrosslinked polyacrylamide (MW 5 × 106) solutions and its separation, on the basis of conformation, from its nicked form is demonstrated in this study. Migration of the supercoiled, nicked circular and linear forms of the plasmid DNA is retarded in proportion to the concentration of uncrosslinked polyacrylamide, the degree of retardation being highest for the nicked circular form. Decreasing the level of supercoiling of the covalently closed circular form by decreasing the concentration of the intercalating dye (ethidium homodimer) shows that the degree of retardation decreases in proportion to the superhelix density.
The Immobiline family: From “vacuum” to “plenum” chemistry
Authors: Chiari Marcella , Righetti Pier Giorgio 
Published: May 2005

AbstractWe list here a total of 17 acrylamido acids and bases as potential buffers and titrants for isoelectric focusing separations in immobilized pH gradients. The chemistry of these compounds is reviewed and general guidelines are given for their proper use. In particular, it is shown that the most delicate compounds are the basic species, since they can undergo several degradation pathways, including: (i) spontaneous hydrolysis to acrylic acid and a diamine; (ii) spontaneous autopolymerization to oligomers and n‐mers; (iii) oxidation to N‐oxides during the persulfate polymerization step. A hydrophobicity scale has been constructed, by partitioning the deprotonated species in water/1‐octanol phases. A scale of resistance to alkaline hydrolysis for the basic acrylamido buffers is also given, followed by general consideration on the structure/stability relationship of these chemicals.
Immobilized pH 2.5–11 gradients for two‐dimensional electrophoresis
Authors: Sinha Pranav , Köttgen Eckart , Westermeier Reiner , Righetti Pier Giorgio 
Published: May 2005

AbstractExtremely wide immobilized pH gradients, pH 2.5–11, for isoelectric separation of complex protein mixtures are described. These pH gradients are theoretically and practically the maximum that can be achieved at present with the available acrylamido buffers and titrants. Conditions are described for reducing conductivity and electroendosmosis in extreme pH ranges. Furthermore, new conditions are described for the separation of proteins in the second dimension. Using this protocol, nearly all the possible cellular products can be separated in one single two‐dimensional map.
Direct detection of carrier ampholytes in immobilized pH gradients using picric acid precipitation
Authors: Knisley Keith A. , Rodkey L. Scott 
Published: May 2005

AbstractA protocol is described for monitoring the heterogeneity of end products of organic syntheses yielding amphoteric molecules containing two or more amino groups. This protocol was found to be a valuable aid in synthesis of carrier ampholytes for specific isoelectric focusing applications. This method does not depend on the ampholytes themselves to dictate the conditions under which they are analyzed. Carrier ampholytes have been found previously to be insoluble in picric acid and the insolubility property was not dependent upon the pI of individual ampholyte species. This insolubility property was exploited in the protocol. Immobilized pH gradients were used to focus the carrier ampholytes. Ampholytes were then visualized in situ by picric acid precipitation. The data shows that the protocol is useful for analyzing the results of chemical manipulations for enhancing the resolution of carrier ampholytes. A direct relationship was shown between carrier ampholyte heterogeneity as demonstrated by this protocol and the resolution of complex protein mixtures in isoelectric focusing gels. Picric acid formed visible precipitates with a variety of organic compounds which contained more than one amino group.
Reliable phenotyping of alpha‐1‐antitrypsin by hybrid isoelectric focusing in an ultranarrow immobilized pH gradient
Authors: Weidinger Sebastian 
Published: May 2005

AbstractGenetically determined phenotypes of the highly polymorphic human alpha1‐antitrypsin were examined by hybrid isoelectric focusing in a narrow immobilized pH gradient. The chosen pH range from 4.45 to 4.75 was useful for identification and classification of the common PI M subtypes and a number of PI variants in the microheterogeneous regions of m6, m7, and m8. A high degree of resolution and an improved sharpness of PI bands was achieved with this excellent technique. It allowed the distinction of a new PI M variant, which has been designated M8, or Mingolstadt, according to the PI nomenclature. The pI difference of this mutant to the slightly cathodically located subtype M3 is approximately 0.001 pH unit. In addition, some common as well as rare phenotypes are presented.
Studies on an apolipoprotein C‐II variant occurring in Caucasians
Authors: Menke‐Möllers Irene , Kurth Jens , Oette Kurt 
Published: May 2005

AbstractApolipoproteins C (apo C‐II, apo C‐III0, apo C‐III1 and apo C‐III2) from delipidated very low density lipoproteins (VLDL) of 522 normo‐ and hyperlipoproteinemic Caucasians were screened by analytical isoelectric focusing. The immobilized pH gradient used was pH 4.0–5.0 with 7 M urea, which raised the apparent pH range to 4.8–5.7. As identified by immunoblotting, six unrelated persons had two major isoforms of apo C‐II, the normal apo C‐II‐1 (which focuses between apo C‐III0 and apo C‐III1) and a variant, designated apo C‐II‐v according to Huff et al. [1], focusing between apo C‐III1 and apo C‐III2 due to a more acidic pI. In narrow pH gradients, apo C‐II‐v can readily be discriminated from the minor isoform, apo C‐II‐2, due to its slightly more basic pI, corresponding to a difference of 0.01 pH units. Neuraminidase treatment did not alter the pI of apo C‐II‐v and on two‐dimensional electrophoresis the molecular weights of apo C‐II‐1 and apo C‐II‐v were indistinguishable. The frequency of apo C‐II‐v was 1.2%. It was the same in males and females and was independent of hypertriglyceridemia. The autosomal codominant inheritance could be demonstrated in the pedigree of one family. Electroblotting of apo C‐II‐1 and apo C‐II‐v onto activated glass fiber sheets, followed by amino acid sequence analysis of the amino terminal ends, revealed an exchange of the amino acid lysine at position 19 by threonine in apo C‐II‐v. The apo C‐II variant is most probably identical to the apo C‐II‐v isolated from a hypertriglyceridemic Caucasian by Huff et al. [1] which has lost the tryptic cleavage site between amino acids 19 and 20. All of our cases were heterozygous. None disclosed hypertriglyceridemia. The binding of apo C‐II‐v to VLDL was reduced. Thus, in homozygosity the quantity of apo C‐II‐v present on VLDL is probably too low for adequate binding and activation of lipoprotein lipase. This may lead to a moderate manifestation of the apo C‐II deficiency syndrome.
The analysis of human serum transferrins with the PhastSystem: Quantitation of microheterogeneity
Authors: van Eijk Hendrik G. , van Noort Willem L. 
Published: May 2005

AbstractThe PhastSystem was used for three different analyses of human serum transferrins (Tf) by isoelectric focusing: (i) The distribution of iron over the two iron binding sites by separation of the four forms: apoTf, TfFec, FeNTf and Fe2Tf by using < 1 μL serum or plasma. The focused bands were visualized by immunoprecipitation and quantified by densitometry (ii) The relative proportions of the diferic sialo‐transferrin fractions in human sera or plasma were determined in a similar way. (iii) The presumed genetic variants can be determined. The methods are easy and reproducible and require much less sample (0.3 μL), time (1 h per run) and antibody (10 μL/sample) than crossed immunoelectrofocusing.
Relative efficiency of molecular sieving in solutions of four polymers
Authors: Chrambach Andreas , Aldroubi Akram 
Published: May 2005

AbstractThe efficiency of size separations of polystyrene sulfate, 120‐1085 nm radius, by molecular sieving in polymer solutions, expressed by the separation efficiency function S = MO dKR(R)/dR Te−KR(R)T [where MO is the mobility in free solution, KR the retardation coefficient, R the geometric mean radius of the particle, KR(R) the retardation coefficient as a function of R, and the polymer concentration is T] increases from methylhydroxypropyl cellulose to polyvinyl alcohol to uncrosslinked polyacrylamide to agarose above its gelling temperature. Separations of DNA, in the size range of 3–21 nm radius, are by at least one order of magnitude more efficient than those of polystyrene sulfate in the size range of 120–1085 nm radius. A plot of S vs. R in the experimental range of T is constructed for the four polymer solutions; this allows one to select optimal media and concentrations for the sieving of particles in the desired range of molecular sizes.
A rapid method to determine the orientation of blunt end ligated polymerase chain reaction products
Authors: Dooley Steven , Seib Thomas , Engel Matthias , Welter Cornelius 
Published: May 2005

AbstractSubcloning of polymerase chain reaction (PCR) fragments is often performed via blunt end ligation after previous Klenow fragment exonuclease treatment. Since insert‐specific primers are at hand from the original amplification step, we used a simple PCR‐based assay to determine the insert orientation of the recombinants. After purification of enriched plasmids, small aliquots were tested performing standard PCR reactions using a plasmid primer directed towards the cloning site and one of the insert specific primers, respectively. Only the distant insert primer yields a PCR product which can be visualized after gel electrophoresis. In this way both the insert orientation in the vector and the correct size of the cloned fragment is determined rapidly without sequencing or restriction fragment analysis.
Analysis of leukotrienes, prostaglandins, thromboxane B2 and their metabolites by capillary isotachophoresis
Authors: Tsikas Dimitrios , Fauler Joachim , Bracht Stefan , Brunner Gorig , Frölich Jürgen C. 
Published: May 2005

AbstractAn analytical capillary isotachophoretic method for the analysis of the eicosanoids leukotriene E4, leukotriene B4, prostaglandins E1 and E2, 6‐keto‐prostaglandin F1α, thromboxane B2, and their metabolites of ω‐ and/or β‐oxidation is described. The method is based on anionic separation and detection by UV absorbance (254 nm) and conductivity and allows simultaneous analysis of the primary compounds and their corresponding major urinary metabolites. The method was applicable to the qualitative and quantitative analysis of prostaglandin E1 in a drug preparation.
A simple physical model for the gel electrophoretic manifestations of sequence‐dependent DNA superstructures
Authors: De Santis Pasquale , Palleschi Antonio , Savino Maria 
Published: May 2005

AbstractA simple physical model of the gel electrophoretic manifestations of sequence‐dependent DNA superstructures is illustrated. The model is based on the calculation of the curvature dispersion as evaluated by integrating the orientational parameters of the base pairs along the chain. Such a quantity is proportional to the straightening activation energy of the curved DNA in the direction of the average orientation of the helical axis and was found to be proportional to the logarithm of the gel electrophoretic retardation. The model is capable of consistently explaining the different electrophoretic manifestations of DNA superstructures, such as the retardation of a large ensemble of multimeric oligonucleotides with different sequences, periodicities, and lengths, and the permutation gel assays as well as the mobility changes consequent to extensive point mutations in a DNA tract.
Analysis of trp repressor‐DNA interactions using gel electrophoresis
Authors: Lewis D. E. A. , Carey Jannette 
Published: May 2005

AbstractQuantitative analysis of the DNA‐binding equilibria of E. coli trp repressor by gel electrophoresis led to reevaluation of our understanding of this complex system. In this review, the data leading to controversy about the trp system are discussed, and our current understanding is presented.
Measurement of binding kinetics using the gel electrophoresis mobility shift assay
Authors: Gerstle Justin T. , Fried Michael G. 
Published: May 2005

AbstractThe gel electrophoresis mobility shift assay is a technique for the qualitative and quantitative analysis of protein‐DNA complexes. The ability to resolve reactants, reaction intermediates and products makes this method particularly well‐suited for the measurement of the assembly and dissociation rates of protein‐nucleic acid complexes. Here we identify conditions that must be met and variations of the technique that are useful for the measurement of reaction rates.
Temperature‐gradient gel electrophoresis for analysis and screening of thermostable proteases
Authors: Sättler Andrea , Riesner Detlev 
Published: May 2005

AbstractThe thermal unfolding of microbial serine proteases was studied by temperature‐gradient gel electrophoresis (TGGE). Conditions for a native polyacrylamide gel electrophoresis were established, and the temperature gradient was applied perpendicularly to the direction of electrophoretic migration. Mobility changes of the protease molecules were indicative for thermally induced conformational changes. The transition temperature was determined with good accuracy. The native and active protease conformation was detected by an activity assay in the temperature‐gradient gel. As a consequence of the typical protease‐autoproteolytic reaction at elevated temperatures, the unfolded protease conformation could not be detected for non‐inhibited, active subtilisin. After inhibition by phenylmethylsulfonyl fluoride (PMSF) the complete structural transition could be followed by TGGE. This transition is “discontinuous”, i.e. the thermal transition is either very slow, compared to the time of electrophoresis, or irreversible, as known for subtilisins from calorimetric data. Inhibition by the strong serine specific inhibitor diisopropyl fluorophosphate (DEP) led to two conformations at low temperature. One conformation is stabilized by 8°C, the other by at least 20°C as compared with PMSF inhibition. The influence of calcium ions on the subtilisin stability was investigated by a series of TGGE under different calcium concentrations. The strong calcium binding site is occupied even without added calcium, occupation of the weak binding site leads to a stabilization of 10°C with a binding constant around 106 M−1. The subtilisin Carlsberg stability could also be investigated in unpurified bacterial culture supernatants. Thus, the method is suitable for screening of thermostable subtilisin mutants directly after expression in a bacterial host. For screening purposes TGGE was modified to a parallel form, which allows investigation of a series of samples in one and the same gel.
Purification of phosphoenolpyruvate carboxylase subunits and isoforms from Vicia faba L. by preparative gel electrophoresis and their detection by enzyme‐linked immunosorbent assay
Authors: Schnabl Heide , Klockenbring Torsten , Huhn Michael , Schulz Margot 
Published: May 2005

AbstractA purification procedure which yields nearly homogenous subunits of stomatal phosphoenolpyruvate carboxylase from epidermal strips of Vicia faba L. is reported. Preparative gel electrophoresis was found to be the most suitable technique for subunit purification. Denatured subunits of the stomatal enzyme in the eluate were immunologically detected by enzyme‐linked immunosorbent assay (ELISA) tests. The enzyme preparation meets all requirements for its use in antibody production.
Structural characterization of blotting membranes and the influence of membrane parameters for electroblotting and subsequent amino acid sequence analysis of proteins
Authors: Eckerskorn Christoph , Lottspeich Friedrich 
Published: May 2005

AbstractVarious blotting membranes were evaluated and correlated with the efficiency of electroblotting and the performance in the sequencing process. Structural parameters including specific surface area, pore size distribution, pore volumes, and permeabilities of different solvents lead to discrimination of the membranes relative to their accessible surfaces and membrane densities. Protein binding capacities as well as protein recoveries in electroblotting correlate with the specific surface areas. Almost quantitative retention of proteins during electroblotting from gels was obtained for membranes with a high specific surface area and narrow pores (Trans‐Blot, Immobilon PSQ, Fluorotrans), whereas membranes with a relatively low specific surface area (Immobilon P, Glassybond) showed reduced recoveries of between 10–20% for the tested proteins. Initial yields and repetitive yields were compared for radioiodinated standard proteins that have been either electroblotted or loaded by direct adsorption. The results showed that the different permeabilities for solutions of the Edman chemistry have a major influence on initial yields. The glass fiber‐based membranes with an extremely low flow restriction produce consistently high initial yields independent of the application mode of the protein (spotted or electroblotted) or the application of the membranes into the cartridge (discs or small pieces). In contrast, the polymeric membranes showed decreasing initial yields with increasing membrane density for spotted and electroblotted proteins. Yields varied considerably when the membranes were applied as discs into the cartridge. This effect could be minimized by cutting the membranes into piecese as small as possible, as demonstrated for electro‐blotted proteins.
Western blotting of basic proteins after nondenaturing electrophoresis in acid conditions using the PhastSystem
Authors: Davril Monique , Ducourouble Marie‐Paule , Van‐Seuningen Isabelle 
Published: May 2005

AbstractElectroblotting of basic proteins was performed from minigels after electrophoresis, under nondenaturing acidic conditions, by using the automated PhastSystem. Depending on the molecular masses of the proteins to be studied, various precast gel media were chosen. The transfer membranes with various types (nitrocellulose and polyvinylidene difluoride) and pore sizes (0.45 and 0.2 μm) were chosen accordingly. For the semidry electric transfer, a simple, discontinuous two‐buffer system was used. The anode solution contained 0.3 M Tris, pH 10.4, and the cathode solution, 40 mM 6‐amino‐n‐hexanoic acid, pH 7.6, with 20% v/v methanol each. The addition of 0.1% sodium dodecyl sulfate (SDS) in the cathode solution facilitated the elution of proteins from the gels and directed the migration of the negative SDS‐protein complexes towards the anode membranes. The transfer conditions following native polyacrylamide gel electrophoresis allowed the visualization of basic proteins, with molecular weights ranging from 29 000 to 5 000, for which isoforms could be resolved and which retained their biological properties.
Protein blotting analysis of large sample numbers
Authors: Kapfhammer Josef P. 
Published: May 2005

AbstractA method is described that allows probing of hundreds of different antibodies on protein blots per day, making it possible to use protein blots for primary screening of hybridoma supernatants. It involves a miniaturization of the protein blots and their attachment to a solid support. This allows a great reduction of the required number of gels and electroblots and brings a drastic simplification of the incubation and washing procedures. Apart from a reduced resolution of closely adjacent bands the resulting mini‐blots are comparable to conventional protein blots in sensitivity and specificity. The procedure has been successfully applied for primary screening of hybridoma supernatants. It can be useful in cases where a monoclonal antibody to an antigen with known molecular weight is required, or in situations where many different antibodies have to be tested in a short time period.
Checkerboard immunoblotting recognizes twenty epitopes among the B subunit proteins of the cholera enterotoxin family
Authors: Qu Zhen‐Hai , Finkelstein Richard A. 
Published: May 2005

AbstractCheckerboard immunoblotting (CBIB) was used to analyze the reactions of a series of monoclonal antibodies with proteins of the cholera enterotoxin (CT) family, including heat labile enterotoxins (LTs) produced by diarrheagenic strains of Escherichia coli and genetically engineered chimeric proteins in which single amino acids of the CT‐B subunit protein or human (H) LT‐B sub‐unit protein were substituted for corresponding residues in porcine (P) LT‐B. The result indicated that there were at least twenty different patterns of reactivity suggesting that there are at least twenty recognizable epitopes among the proteins studied. An epitope which includes Ala46 appears to be particularly important. This epitope is common to CT and H‐LT but not P‐LT, and the epitope is not blocked by the Gml ganglioside. Human convalescent sera react with this epitope. CBIB is a versatile technique for epitope analysis.
Rapid electroelution of two‐dimensionally separated protein mixtures: Its use in in vitro assays of T cell activities
Authors: Gulle Heinz , Kaufmann Stefan H. E. , Moriarty Kevin M. 
Published: May 2005

AbstractA recently developed electroelution method for separated mixtures of proteins and its application in vaccine research were investigated. The method combines the high resolution power of two‐dimensional gel electrophoresis with the advantage of direct probing of separated proteins with viable cells. An electroelution time of only 30 min was sufficient for complete protein transfer, as shown by Coomassie Brilliant Blue and silver staining. Inclusion of sodium dodecyl sulfate (SDS) into the electrophoresis buffer for the second dimension considerably improved the separation capacity. Furthermore, because of the low concentration of SDS (0.03%) no deleterious effects on the cells were seen. It was shown that T lymphocytes from cattle vaccinated with dead M. bovis BCG responded to numerous mycobacterial protein antigens, whereas unvaccinated control animals showed no, or very weak, responses. A comparison of T cell proliferation profiles obtained with different protein separations demonstrated the reproducibility of the method.