bluuebunny/crossref_metadata_2025 huggingface dataset:
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

Circular 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

We 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

Extremely 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

A 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

Genetically 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

Apolipoproteins 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

The 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

The 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

Subcloning 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

An 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

A 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

Quantitative 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

The 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

The 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

A 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

Various 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

Electroblotting 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

A 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

Checkerboard 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

A 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.
Immunoblotting of Yersinia plasmid‐encoded released proteins: A tool for serodiagnosis
Authors: Cremer Josef , Putzker Michael , Faulde Michael , Zöller Lothar
Published: May 2005

The plasmid‐encoded, released proteins (RPs) of Yersinia enterocolitica seroypes 09 and 03 were separated by sodium dodecyl sulfate (SDS)‐pore gradient gel electrophoresis. The RP‐patterns of both serotypes proved to be identical. Five major proteins of Mr 27000, 34700, 35600, 45800, and 46800 were detected. Spontaneously plasmid‐cured derivatives of the two serotypes lost the feature of protein release. By immunoblotting of RP with sera from patients suffering from acute Yersinia infections, specific and reproducible band patterns were obtained. Laser scan densitometry was applied to record the immunoreactions quantitatively. Predominant bands were detected at an Mr of 34700 and 35600. IgA and IgM antibodies appeared as acute‐phase markers rapidly decreasing in the reconvalescent phase. In contrast, immunoblots of patients with supposed chronic yersiniosis were characterized by a persisting IgA and elevated IgG reactivity. The application of RP as diagnostic antigens proved to be advantageous because they are naturally separated from cross‐reacting proteins, common to pathogenic and nonpathogenic strains of Yersinia enterocolitica and Y. pseudotuberculosis.
On the efficiency of methylene blue versus persulfate catalysis of polyacrylamide gels, as investigated by capillary zone electrophoresis
Authors: Caglio Silvia , Righetti Pier Giorgio
Published: May 2005

The efficiency of a novel method of photopolymerization, based on photoinitiating the reaction with methylene blue (MB), in presence of a redox couple (sodium toluenesulfinate and diphenyliodonium chloride), vs. the conventional persulfate ‐ N,N,N′,N′ ‐tetramethylethylenediamine redox couple was investigated as a function of different effectors in solution. Oxygen dissolved in the gelling mixture strongly quenches persulfate catalysis, while leaving essentially unaltered the process initiated by photopolymerization. On the contrary, the presence of 8 M urea substantially accelerates a persulfate‐driven reaction, boosting the conversion of monomers to near completion (> 98%) while leaving the photopolymerization process largely unaffected. Polyacrylamide polymerization has also been performed in a number of hydroorganic solvents (all in a 50 : 50 v/v ratio): dimethyl sulfoxide (DMSO), tetramethyl‐urea, formamide and dimethylformamide. In all cases, the persulfate‐catalyzed reaction was strongly quenched and even completely inhibited (in DMSO), whereas the photopolymerization process was essentially unaffected by any of these organic solvents. The reaction kinetics of the methylene blue‐driven reaction could not be ameliorated when admixing an anionic dye (e.g., eosin Y) to the cationic MB, even when amply changing their molar ratios. Thus, it appears that photocatalysis with MB (and the redox couple sodium toluene‐sulfinate and diphenyliodonium chloride) is a unique process, proceeding at optimum rate under the most adverse conditions, completely insensitive to any kind of positive and negative effectors and able to ensure at least 95% monomer conversion under the standard conditions of 1 h reaction time at room temperature. Due to the fact that the MB system is also unaffected by the pH of the gelling solution (in the pH 4–10 interval), it would appear that such a system is largely superior to any other catalyst mixture so far proposed in the field of polyacrylamide catalysis.
Native polyacrylamide gel electrophoresis of membrane proteins: Glutaminase detection after in situ specific activity staining
Authors: Aledo Juan C. , Gómez‐Biedma Simón , Segura Juan A. , Molina Manuel , de Castro Ignacio Núñez , Márquez Javier
Published: May 2005

A new procedure for the analysis and detection of phosphate‐activated glutaminase (EC 3.5.1.2) by native electrophoresis has been developed. The method is based on the in situ detection of glutaminase activity in two different systems of native polyacrylamide gradient gels, containing 3‐(3‐cholamidopropyl)‐dimethyl‐ammonio‐1‐propane sulfonate (CHAPS) or Triton X‐100 as nondenaturant detergents. Crude Triton X‐100 extracts of mitochondria were resolved by electrophoresis. The enzyme was specifically revealed by incubation of the gel with glutamine and coupling the oxidation of the glutamate formed to the reduction of a tetrazolium dye, in the presence of glutamate dehydrogenase trapped in a 1% agar solid overlay. Both Ehrlich ascitic cell and mouse kidney glutaminases were resolved by native electrophoresis and specifically detected with the activity staining. Moreover, the redox‐cycling staining was tested in solution, showing linearity with the amount of glutamate or glutaminase activity present. The method described could be a useful tool for native polyacrylamide gel electrophoresis of membrane proteins.
Apolipoprotein (a): A comparison of isoforms identified by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis or by sodium dodecyl sulfate‐agarose gel electrophoresis
Authors: Craig Wendy Y. , Poulin Sue E. , Ledue Thomas B. , Kamboh M. Ilyas
Published: May 2005

Lipoprotein(a) resembles low density lipoprotein in structure, except that a unique apolipoprotein (apo), apo(a), is linked to apo B‐100. Variations in the number of sequence repeats in the apo(a) gene give rise to a range of isoforms. Depending on the method used, 6–30 apo(a) isoforms have been observed; however, the correspondence of these different isoforms has not been reported, making between‐study comparisons difficult. In the present study we address this question by characterizing the apo(a) phenotypes of 48 sera using two previously reported separation methods, sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE, 3–12% gels) and SDS‐agarose gel electrophoresis. In addition, the molecular weight of each isoform was estimated using haptoglobin 2–2 polymers as molecular weight standards. Among the 48 sera, 15 distinct apo(a) isoforms were separated by SDS‐PAGE and 28 by SDS‐agarose gel electrophoresis. There was excellent correlation between the two nomenclature systems (r = −0.97, p < 0.001, by rank correlation), and the ranges were totally overlapping, with the same two isoforms being identified as the largest and smallest by either method. The apparent molecular mass range for the isoforms was 294–624 kDa, which is in close agreement with the theoretical molecular mass range of 238–643 kDa, calculated from the sequence and carbohydrate content of recombinant apo(a). The disparity in number of isoforms between methods was expected, due to the poorer separation of apo(a) by SDS‐PAGE; 3.1 ± 1.7 (median, 2.0) SDS‐agarose isoforms were combined for each SDS‐PAGE isoform. The present study demonstrates that the nomenclature systems for apo(a) isoforms separated by SDS‐PAGE or by SDS‐agarose gel electrophoresis are well correlated mathematically and encompass the same size range; however, the better resolution of SDS‐agarose electrophoresis suggests that it is the method of choice for apo(a) phenotyping. As further apo(a) isoforms are identified, it will be important to address the question of a standardized nomenclature, in order to facilitate between‐study comparisons.
Salivary deoxyribounuclease I polymorphism separated by polyacrylamide gel‐isoelectric focusing and detected by the dried agarose film overlay method
Authors: Tenjo Etsuko , Sawazaki Kazumi , Yasuda Toshihiro , Nadano Daita , Takeshita Haruo , Iida Reiko , Kishi Koichiro
Published: May 2005

Isoelectric focusing of whole saliva samples on polyacrylamide gels (pH 3.5–5), followed by dried agarose film overlay detection, was employed to determine the type of salivary deoxyribonuclease I (DNase I). Since this detection method had not only a high sensitivity, but also a high band resolution, it was possible to determine DNase I types from saliva samples of 2–5 μL. Pretreatment of saliva samples with neuraminidase simplified the isozyme pattern and enhanced the sensitivity. The DNase I types in all 30 saliva samples showed a good correlation with the types found in the corresponding blood, semen, and urine samples. This preliminary trial indicates that DNase I typing from saliva samples is a new and promising method for individualization of casework samples in the field of forensic biology.
Nonenzymatic extraction of cells from clinical tumor material for analysis of gene expression by two‐dimensional polyacrylamide gel electrophoresis
Authors: Franzén Bo , Linder Stig , Okuzawa Ken , Kato Harabumi , Auer Gert
Published: May 2005

We have compared different methods of preparation of malignant cells for two‐dimensional electrophoresis (2‐DE). We found all methods using fresh tissue to be superior compared to methods using frozen tissue. Our results indicate that nonenzymatic methods of preparation of tumor cells, including fine needle aspiration, scraping and squeezing, have advantages over methods using enzymatic extraction of cells. Nonenzymatic methods are rapid, appear to reduce loss of high molecular protein species, and alleviate the necessity of separating viable and nonviable cells by Percoll gradient centrifugation. Using these techniques, high‐quality 2‐DE maps were derived from tumors of the lung and breast. In the resulting polypeptide patterns, heat shock proteins, non‐muscle tropomyosins and intermediate filament were identified. We conclude that nonenzymatic extraction of malignant cells from fresh tumor tissue improves the possibilities that these techniques may be useful in clinical diagnosis.
Studies on 16 kDa selenium‐containing proteins enriched by means of preparative electrophoresis
Authors: Kyriakopoulos Antonios , Kalcklösch Margrit , Weiß‐Nowak Christian , Behne Dietrich
Published: May 2005

By in vivo labeling with [75Se]selenite and separation of the proteins in the tissue homogenates by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), several selenium‐containing proteins or protein subunits were detected in rat tissues (liver, lung, spleen and prostate). Their distribution among the cell components was investigated after fractionation by means of differential centrifugation. The selenium‐containing proteins in the 16 kDa range were found to be mainly membrane‐bound. By two‐dimensional electrophoresis they were resolved into three labeled spots, two with the same relative molecular mass and pI values of about 4.8 and 5.0 and the third with a slightly lower molecular mass and a pI of 4.8. For further investigation they were concentrated and separeted from the other labeled compounds by SDS‐PAGE using preparative flow‐through electrophoresis.
Our obsession with high resolution in gel electrophoresis: Does it necessarily give the right answer?
Authors: Wrigley Colin W. , Gupta Ram B. , Bekes Ference
Published: May 2005

Poor resolution of protein zones in an electrophoretic pattern may not necessarily be the result of poor technique. The example is given of the ‘streak material’, extracted from wheat flour, now recognised to be aggregated subunits of glutenin. The size distribution of the aggregated glutenin ‘streak’ is the key to elucidating the functional properties of wheaten dough. A stepped‐layer gel technique has been devised to quantitate the proportions of aggregated glutenin in specific size groupings.
Control of electrohydrodynamic distortion of sample streams in continuous flow electrophoresis using oscillating fields
Authors: Heller Christoph , Limat Laurent , Sergot Philippe , Viovy Jean Louis
Published: May 2005

Continuous flow electrophoresis is a method to separate ions contained in a sample continuously injected into a laminar flow of electrolyte as a cylindrical stream. Usually, the sample is more conductive than the electrolyte, and the charges created at the sample‐electrolyte interface lead to electrohydrodynamic distorsions which reduce the separation power of this technique. We demonstrate theoretically that the rate of electrohydrodynamic distorsion of a cylindrical sample stream can be reduced to zero, by superimposing to the AC field responsible for the separation of a DC field transverse to it and to the flow direction, with an appropriate frequency, and an effective strength equal to that of the DC field. Using a continuous flow electrophoresis chamber, in which such a field is produced using capacitive electrodes, the major predictions of the theory are confirmed. In particular, it is shown that a sample stream more conductive than the carrying electrolyte, which was seriously deformed in the absence of a transverse AC field, recovers its cylindrical shape in presence of the field. The implications of this discovery for the separating power of continuous‐flow electrophoresis are discussed.
Centrifuge‐blotting of proteins after separation by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis
Authors: Hermansen Leonila F. , Pedersen Øyvind , Sletten Knut
Published: May 2005

A protein transfer method which allows elution and immobilization of polypeptides onto a polyvinylidene difluoride (PVDF) membrane has been developed. The protein band in a gel is eluted by centrifugation. The centrifugeblotting procedure involves the following steps: (i) visualization of the protein in a sodium dodecyl sulfate (SDS)‐polyacrylamide gel with 1 M KC1, (ii) excision of the protein band and equilibration for 15 min in a solution of 0.05% SDS/5% methanol/0.02% dithiothreitol in distilled water, (iii) placing the gel piece in direct contact with the PVDF membrane in the receptacle, (iv) centrifugation at 3000 g for 1 h. A 10 kDa cut‐off dialysis membrane is placed beneath the PVDF membrane to retain nonimmobilized protein. The N‐terminal sequence of the immobilized protein on the PVDF membrane was determined. For proteins with a molecular mass less than 30 kDa, an overall yield between 10%–30% has been obtained.
Salivary protein interactions with oral bacteria: An electrophoretic study
Authors: Newman Frances , Beeley Josie A. , MacFarlane T. Wallace , Galbraith Julie , Buchanan Luise
Published: May 2005

The relatively low levels of human salivary proteins in whole saliva as compared to the ductal secretions may be related to their interaction with oral bacteria. These interactions are thought to play an important role in the microbial colonisation of the mouth, and salivary proteins adsorbed to oral surfaces have been implicated in adherence. In this study we attempted to investigate the prevalence of interactive strains by screening a range of oral bacteria. Parotid saliva was incubated with bacterial suspensions and consequent alterations to the salivary protein profile determined by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). A number of salivary components were found to be preferentially removed, particularly by those species known to be primary colonisers of clean tooth surfaces.
A nonlinear wide‐range immobilized pH gradient for two‐dimensional electrophoresis and its definition in a relevant pH scale
Authors: Bjellqvist Bengt , Pasquali Christian , Ravier Florence , Sanchez Jean‐Charles , Hochstrasser Denis
Published: May 2005

A new nonlinear immobilized pH gradient (IPG) is proposed as the first dimension for two‐dimensional electrophoresis. In comparison to conventional carrier ampholyte techniques, it offers better resolution and greater reproducibility whilst allowing application of higher protein loads. Furthermore, we have checked and supplemented existing data on pK values for the immobilized groups in the presence of 8M urea. This is necessary for pH gradients to be defined in a pH scale relevant to the focusing conditions such that spot positions can be related to amino acid compositions. The data will allow definition of pH scales for the temperature range 10–25°C and for a pH range covering the major part of the nonlinear pH gradient. With the latter, focusing positions are neither influenced by urea concentration nor by the choice or the concentration of detergent or carrier ampholyte. Temperature is the only parameter affecting focusing reproducibility and here any changes in focusing positions can be related to the amino acid compositions of peptides.
Evolution of coxsackie B virus during in vitro persistent infection: Detection of protein mutations using two‐dimensional polyacrylamide gel electrophoresis
Authors: McLaren John , Argo Evelyn , Cash Phillip
Published: May 2005

Serotype 5 coxsackie B virus (CBV5) can establish in vitro persistent infections in human rhabdomyosarcoma (RD) cells. This paper describes the characterisation of the virus released from the persistently infected RD cell line designated piRD‐3673. Although infectious virus was released for 42 sequential passages of piRD‐3673 cells, no gross virus‐specific cytopathic effect was detected when the cells were examined by light microscopy. Two‐dimensional polyacrylamide gel electro‐phoresis was used to compare the virus released from piRD‐3673 cells with the CBV5 isolate (CBV‐3673) used to initiate the persistent virus infection. Two of the virus intracellular proteins (apparent molecular weights 33 000 and 39 000, designated p33 and p39, respectively) increased in their net basic charge for the virus released from piRD‐3673 cells compared to CBV‐3673; a reduction in the apparent molecular weights of p33 and p39 was also observed. The charge alteration for both p33 and p39 was a two‐stage process, the accumulative effect of which resulted in p33 increasing in pI from 6.14 to 6.53 and p39 increasing in pI from 6.29 to 6.63. The first mutation of p33 and p39 occurred between passages 7 and 10 of piRD‐3673 cells and affected both the charge and apparent molecular weight of these two proteins. The second mutation at passage 15 of piRD‐3673 cells caused only a change in the charge of p33 and p39. Two other virus proteins (p54 and p75) showed no evidence of mutation over the same passage history of piRD‐3673 cells. The virus released from piRD‐3673 cells also differed from CBV‐3673 by two further criteria, a reduction in plaque‐forming efficiency in HEp‐2 cells and increased virus replication in RD cells. These data on virus evolution are discussed in relation to the maintenance of persistent CBV infections and the presence of naturally occurring CBV variants.
On the kinetics of photopolymerization: A theoretical study
Authors: Lyubimova Tatyana , Righetti Pier Giorgio
Published: May 2005

In dye‐sensitized polymerization, some paradoxical phenomena have been found, such as an anomalous decrease of reaction rate with decrementing thicknesses of the gelling layers. By mathematical modelling, and experimental verification, it has been found that high incorporation efficiencies (> 95% conversion of monomers into the growing polymer) can only be obtained at the correct levels of dye in the gelling solution and by using the proper light intensity. Paradoxically, if levels of sensitizer or incident light intensities are too high, the rate of photosen‐sitizer consumption is too high, as compared with the rate of monomer incorporation, so that dye depletion occurs prior to chain elongation, and the reaction suddenly comes to a stop. In methylene‐blue catalysis, the curve correlating the monomer incorporation with the catalyst concentration exhibits a maximum, indicating poor conversions both below and above a critical catalyst level. When correctly used, photopolymerization still offers unique advantages over chemical (persulfate) polymerization, such as absence of oxidizing conditions and control at the onset of reaction, while guaranteeing high conversion efficiencies (> 95%).
Use of fluorogenic substrates to visualize trypsin and chymotrypsin inhibitors after electrophoresis
Authors: Weder Jürgen K. P. , Haußner Kristine , Bokor Maria V.
Published: May 2005

Fluorogenic substrates were tested as a means of increasing both the sensitivity and the selectivity of trypsin and chymotrypsin inhibitor detection after electrophoretic separation. Out of six substrates applied to cellulose acetate membranes, N a‐benzyloxycarbonyl‐L‐arginine‐4‐methylcoumarinyl‐7‐amide (Z‐Arg‐MCA) and benzylxycarbonyl‐glycyl‐glycyl‐L‐arginine‐4‐trifluoromethylcoumarinyl‐7‐amide (Z‐Gly‐Gly‐Arg‐TFMCA) were found to be suitable for trypsin, and L‐alanyl‐L‐alanyl‐L‐phenylalanine‐4‐methylcoumarinyl‐7‐amide (Ala‐Ala‐Phe‐MCA) was suitable for chymotrypsin. A procedure to detect trypsin and chymotrypsin inhibitors, and to discriminate between them, was developed. After electrophoresis, slab gels were first incubated with the enzyme (bovine trypsin, bovine chymotrypsin, or human duodenal juice) at 37°C, and then covered with the respective substrate membrane and incubated at room temperature while being observed under UV light. Dark blue inhibitor bands on a light‐blue‐fluorescent background were obtained with Z‐Arg‐MCA/trypsin and Ala‐Afa‐Phe‐MCA/chymotrypsin, whereas Z‐Gly‐Gly‐Arg‐TFMCA/trypsin resulted in dark inhibitor bands on a fluorescent green background. The “inhibitor overlay membrane technique” (IOM technique) was used after polyacrylamide gel isoelectric focusing with carrier ampholytes and immobilized pH gradients, pore‐gradient polyacrylamide gel electrophoresis, and sodium dodecyl sulfate pore‐gradient polyacrylamide gel electrophoresis.
Restriction landmark genomic scanning method and its various applications
Authors: Hayashizaki Yoshihide , Hirotsune Shinji , Okazaki Yasushi , Hatada Izuho , Shibata Hideo , Kawai Jun , Hirose Kenji , Watanabe Sachihiko , Fushiki Shinji , Wada Seiji , Sugimoto Toshikado , Kobayakawa Kiyoshi , Kawara Tsutomu , Katsuki Motoya , Shibuya Tohru , Mukai Tsunehiro
Published: May 2005

We have developed a new genome scanning method (restriction landmark genomic scanning (RLGS)), based on the new concept of using restriction enzyme sites as landmarks. RLGS employs direct end labeling of the genomic DNA digested with a restriction enzyme and two‐dimensional electrophoresis with high‐resolution. Its advantages are: (i) high‐speed scanning ability, allowing simultaneous scanning of thousands of restriction landmarks; (ii) extension of the scanning field using different kinds of landmarks in an additional series of electrophoresis; (iii) application to any type of organism because of direct‐labeling of restriction enzyme sites and no hybridization procedure; and (iv) reflection of the copy number of the restriction landmark by the spot intensity which enables distinction of haploid and diploid genomic DNAs. The RLGS method has various applications because it can be used to scan for physical genomic DNA states, such as amplification, deletion and methylation. The copy number of the locus of a restriction landmark can be estimated by the spot intensity to find either an amplified or deleted region. The methylation state of genomic DNA can also be discovered by use of a methylation‐sensitive restriction enzyme sites as a restriction landmark (restriction landmark genomic scanning for screening methylated sites, RLGS‐M). This article introduces the basic principle of RLGS and its applications to the analysis of cancer, mouse mutant DNAs and tissue‐specific methylation, showing the usefulness of RLGS for a variety of biological fields.
Two‐dimensional gel selection of protein binding sites on DNA
Authors: Boffini Andrea , Prentki Pierre
Published: May 2005

We have developed a gel electrophoretic approach for visualizing and cloning protein binding sites from complete genomes. This system consists of a simple two‐dimensional band shift, in which protein‐DNA complexes are retarded in the first dimension, performed at low temperature, and disrupted in the second dimesion, performed at high temperature. We present here results obtained with the integration host factor (IHF) and cAMP receptor protein (CRP) proteins of Escherichia coli, and discuss some of the important methodological aspects of the technique.
Pulsed field agarose gel electrophoresis in the study of morphogenesis: Packaging of double‐stranded DNA in the capsids of bacteriophages
Authors: Serwer Philip , Hayes Shirley J. , Moreno Elena T. , Louie Donna , Watson Robert H. , Son Marjatta
Published: May 2005

To understand how comparatively simple macromolecular components become biological systems, studies are made of the morphogenesis of bacteriophages. Pulsed field agarose gel electrophoresis (PFGE) has contributed to these studies by: (i) improving the length resolution of both mature, linear, double‐stranded bacteriophage DNAs and the concatemers formed both in vivo and in vitro by the end‐to‐end joining of these mature bacteriophage DNAs, (ii) improving the resolution of circular conformers of bacteriophage DNAs, (iii) improving the resolution of linear single‐stranded bacteriophage DNAs, (iv) providing a comparatively simple technique for analyzing protein‐DNA complexes, and (v) providing a solid‐phase quantitative assay for all forms of bacteriophage DNA; solid‐phase assays are both less complex and more efficient than liquid‐phase assays such as rate zonal centrifugation. Conversely, studies of bacteriophages have contributed to PFGE the DNA standards used for determining the length of nonbacteriophage DNAs. Among the solid‐phase assays based on PFGE is an assay for excluded volume effects.