bluuebunny/crossref_metadata_2025 huggingface dataset:
Wheat cultivar identification by a totally automatic softlaser scanning densitometry and computer‐aided analysis of protein electropherograms
Authors: Autran Jean‐Claude , Abbal Philippe
Published: May 2005

A computer‐aided analysis based on soft‐laser densitometry of electrophoretic patterns is described. The system has the potential to automatically identify wheat cultivars from normalized sodium dodecyl sulfate – polyacrylamide gel electropherograms. The principle emcompasses autocalibration of the relative mobilities of components of the unknown pattern using standards of Mr that bracket each pattern on the pictures, automatic identification of peaks and baseline correction, and simplified estimation of the peak intensities. The algorithm provides a normalized array that can be automatically compared with a library of standard cultivar patterns in order to determine those having the highest similarity percentage. The outputs of the program are (1) relative similarity plots which show the percentage of similarity between the unknown cultivar to that of each of the common standard cultivars put in a pattern library, (2) the names of cultivars in the library having the most similar patterns and (3) computer‐generated electrophoretic graphics of these cultivars. This system is not intended to identify closely related cultivars but it is particularly recommended for cultivar identification without well‐trained personnel, familiar with band location and nomenclature.
Proline hydroxylation alters the electrophoretic mobility of pulmonary surfactant‐associated protein A
Authors: Phelps David S. , Floros Joanna
Published: May 2005

Studies from several laboratories involving amino acid analysis and sequencing of the Mr 35 000 pulmonary surfactant‐associated proteins (SP‐A) have detected hydroxyproline residues. These residues are present in a region with a collagen‐like sequence that has been revealed by direct amino acid sequencing and from the deduced amino acid sequence of the cDNA clones coding for SP‐A. We treated human lung tissue with tunicamycin to block N‐glycosylation and with 2,2‐dipyridyl to inhibit the hydroxylation of proline residues. The SP‐A synthesized under these conditions showed a shift in apparent molecular weight to 27 000 and 29 000 compared to 29 000 and 31 000 for SP‐A synthesized in the presence of tunicamycin alone. Dipyridyl treatment alone caused an alteration in electrophoretic mobility similar to that seen with tunicamycin, although this was more difficult to evaluate since changes in molecular weight due to glycosylation occurred under these conditions. These results indicate that proline hydroxylation in the collagen‐like portion of SP‐A decreases its electrophoretic mobility.
Improvement and simplification of low‐background silver staining of proteins by using sodium dithionite
Authors: Rabilloud Thierry , Carpentier Gilles , Tarroux Philippe
Published: May 2005

High sensitivity and low background, the attractive characteristics of the procedure of Blum et al., Electrophoresis 1987, 8, 93–99, for silver staining of proteins in polyacrylamide gels have been improved by sensitizing the gels with sodium dithionite instead of sodium thiosulfate and by equilibration in water after fixation and prior to sensitization. These modifications decrease the background and allow for a longer development period, which in turn increases sensitivity and color contrast. In addition, the colors of the spots are shifted toward colder tones when compared with the original method.
Determination of molecular mass, stokes' radius, frictional coefficient and isomer‐type of non‐denatured proteins by time‐dependent pore gradient gel electrophoresis
Authors: Rothe Gunter M.
Published: May 2005

Molecular mass, Stokes' radius, frictional coefficient and isomer‐type of non‐denatured proteins can be obtained by time‐dependent gradient gel electrophoresis by evaluating the resulting data using a two‐step mathematical procedure. Provided a histochemical staining procedure is available to locate the position of an enzyme in the gel, crude cell extracts can be used for estimating their molecular size properties. The computation of molecular properties of non‐denatured proteins is demonstrated for isozymes of aspartate aminotransferase (EC 2.6.1.1), peroxidase (EC 1.11.1.42) and glucose‐6‐phosphate dehydrogenase (EC 1.1.1.49) from current‐year needles of spruce. The resulting data as well as those which were calculated for esterase (EC 3.1.1.1), glutamate dehydrogenase (EC 1.4.1.4), isocitrate dehydrogenase (EC 1.1.1.42) and shikimate dehydrogenase (EC 1.1.1.25) are in accordance with those reported in the literature. The method described may be applied to various scientific areas such as genetics or environmental pollution. It could be shown here that current‐year needles of injured spruce (damage class 3) contained two more peroxidase isozymes and one more glucose‐6‐phosphate dehydrogenase isozyme than those from non‐injured trees. These differences may mark two genotypes of spruce of different susceptibilities towards present‐day air and soil pollutants.
N‐terminal and internal sequence determination of microgram amounts of proteins separated by isoelectric focusing in immobilized pH gradients
Authors: Aebersold Ruedi H. , Pipes Gary , Hood Leroy E. , Kent Stephen B. H.
Published: May 2005

Isolation of microgram amounts of proteins and submicrogram quantities of peptides in a form suitable for sequence analysis is a key step in high sensitivity protein sequencing technology. Recently, methods have been developed which allow the direct, high yield, recovery of microgram amounts of sequenceable proteins separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis or two‐dimensional gel electrophoresis. In the present publication, we describe an extension of these methods to obtain N‐terminal or internal sequence information from proteins separated by isoelectric focusing in polyacrylamide gels containing immobilized pH gradients. For N‐terminal sequence analysis, separated proteins were electrophoretically transferred (electroblotted) onto chemically‐modified glass fiber sheets, a support compatible with Edman degradation chemistry. Transferred protein bands were detected on the support, cut out and directly inserted into the cartridge of a gas‐phase protein sequenator. For internal sequence analysis, separated proteins were electrophoretically transferred onto nitrocellulose. Protein bands were detected by staining, cut out and the proteins subjected to enzymatic digestion directly on the support. The resulting cleavage fragments (peptides) were released into the supernatant where they were recovered and separated by narrow‐bore reversed‐phase high performance liquid chromatography for sequence analysis. The potential of this methodology is illustrated by the comparative peptide mapping of isoforms of bovine carbonic anhydrase.
Microheterogeneity of human serum transferrin: A biological phenomenon studied by isoelectric focusing in immobilized pH gradients
Authors: de Jong Gerard , van Eijk Hendrik G.
Published: May 2005

The heterogeneity of human transferrin results from (i) differences in iron content, (ii) genetic polymorphism and (iii) differences in the carbohydrate moiety. This article primarily deals with the last phenomenon, the microheterogeneity of human transferrin. Owing to the comparatively simple carbohydrate structure of human transferrin and the high resolving power of isoelectric focusing in immobilized pH gradients, microheterogeneous forms of transferrin can be separated. Differences between samples can be quantitated by crossed immunoelectrophoresis. Examples of the differences between the microheterogeneity patterns of transferrin in several biological fluids and the changes that can be observed in diseases such as rheumatoid arthritis, idiopathic hemochromatosis and Kahler's disease are presented. Special attention has been focused on changes occurring during pregnancy.
Two‐dimensional electrophoresis as a tool for structural and genetic studies of seed proteins from Poaceae and Fagaceae
Authors: Garcia‐Olmedo Francisco , Salcedo Gabriel , Aragoncillo Cipriano , Sánchez‐Monge Rosa , Collada Carmen , Gómez Luis
Published: May 2005

The application of two‐dimensional electrophoretic procedures to structural and genetic studies of seed proteins from Poaceae (including the cultivated cereals) and Fagaceae is described. The following related problems have been considered: covalent and non‐covalent association of protein subunits in multiple oligomeric structures; chromosomal locations of genes encoding seed proteins; quantitation of gene products in relation to gene expression and regulation; purification of protein components to study their homology relationships and in vitro activities; evolutionary and phylogenetic relationships; identification of genetic stocks. Isoelectric focusing, pore‐gradient electrophoresis, electrophoresis at different pH's, are among the separation procedures used in the first dimension, whereas sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and starch‐gel electrophoresis at acid pH have been the preferred second‐dimensional methods. Dissociating conditions (sodium dodecyl sulfate, Nonidet P‐40, or urea) and reducing conditions (2‐mercaptoethanol) have been used when required.
Use of electrophoretic techniques in determining the composition of seed storage proteins in alfalfa
Authors: Krochko Joan E. , Bewley J. Derek
Published: May 2005

Holoprotein molecular weights and polypeptide composition can be determined for complex mixtures of oligomeric proteins using two‐dimensional electrophoretic techniques. The variety of two‐dimensional analyses presented here is a reflection of the general usefulness of each method for the identification and characterization of the different classes of seed storage proteins in alfalfa. These techniques can be applied to studies of storage proteins in other seeds as well as non‐seed storage proteins. The major seed storage proteins in alfalfa are medicagin (a legumin‐like globulin), alfin (a vicilin‐like globulin) and a family of lower molecular weight albumins (LMW1–3). These comprise 30 %, 10 %, and 20 %, respectively, of the total extractable protein from cotyledons of mature seeds. Alfin is a heterogeneous oligomeric protein (Mr ∼ 150 000) composed of polypeptides ranging in size from Mr 14 000 to 50 000 (α1‐α6; 50 000, 38 000, 32 000, 20 000, 16 000 and 14 000, respectively). Medicagin is also a high molecular weight oligomeric protein, but requires high concentrations of salt for solubilisation. It is comprised of a family of individually distinct subunits, each composed of an acidic polypeptide (A1–A9; Mr 49 000 to 39 000) linked via disulphide bond(s) to a basic polypeptide (B1, B2, B3; Mr 24 000, 23 000 and 20 000, respectively). This pairing is highly specific and two families are recognizable on the basis of the B polypeptide (B3 or B1 /B2). Subunits (Mr ∼ 50 000–65 000) are assembled as trimers (8S) or larger oligomers (12S–15S) in mature seeds. The lower molecular weight albumins (LMW1–3) are acidic (pI < 6), and consist of sets of disulphide‐bonded polypeptides (Mr 15 000 and 11 000).
Improvement of electrophoretic transfer by casting acrylamide gels on a cellophane sheet
Authors: Abbal Michel , Julie Claudine
Published: May 2005

Electrophoretic transfer of protein after isolectric focusing using a polyacrylamide gel of less than 0.5 mm is difficult if the gel is backed to an electrically nonconductive casting support, such as glass plate or plastic films. By casting the gel on a cellophane sheet, it is not necessary to remove the gel from the support prior to electrophoretic transfer. The use of a cellophane support does not alter the quality of the final pattern.
Fully automated capillary isotachophoresis of proteins
Authors: Manabe Takashi , Yamamoto Hideko , Okuyama Tsuneo
Published: May 2005

An apparatus for fully automated capillary isotachophoresis was constructed. A commercial apparatus (Shimadzu IP‐2A) was modified in the electrolyte pumping system and the flow lines were simplified. An automatic sampler was used for sequential sampling. The equipment, pumps, the sampler, a high‐voltage DC power supply, and a recorder, were controlled by a system controller which comprises a microcomputer and a BASIC program for time‐control of the equipments. The apparatus was successfully used for the automated sequential analysis of human serum proteins. Forty serum samples were analyzed within 17 h without manual operation and for each sample the serum proteins were resolved into about twenty UV peaks or shoulders.
Transverse alternating field electrophoresis and applications to mammalian genome mapping
Authors: Gardiner Katheleen , Patterson David
Published: May 2005

The transverse alternating field electrophoresis system is a pulsed field gel apparatus that has been used to separate DNA molecules that range in size from a few thousand to approximately 7 million base pairs. This apparatus uses a vertical gel and a simple electrode arrangement to produce electric fields that are uniform across all lanes of the gel. The velocity of identical molecules does not vary from lane to lane, and hence there is no distortion in the paths of the DNA. The performance of this system is illustrated here using the chromosomes from S. pombe and S. cerevisiae, and restriction enzyme digested mammalian DNA. The mobility of molecules up to 1100 kilobase pairs is linear with size and can be accomplished in overnight runs. Resolution of very large molecules requires electrophoresis for several days, but molecules from 200 to 7000 kilobase pairs can be separated on a single gel. This electrophoresis system has been used extensively in the construction of a physical map of human chromosome 21, and examples of this application are discussed.
Gel electrophoresis method for quantitation of gamma ray induced single‐ and double‐strand breaks in DNA irradiated in vitro
Authors: Chen Chun‐Zhang , Sutherland John C.
Published: May 2005

We describe a method based on gel electrophoresis for the quantitation of strand breaks in DNA and demonstrate its application to the measurement of single‐ and double‐strand breaks formed by γ‐rays for DNA irradiated in vitro. For single‐strand breaks, our data span the dose range from 0.1 to 1 Gy, while for double‐strand break doses were from 3 to 15 Gy. In agreement with results obtained using other techniques, we find that the dose response function for single‐strand breaks is linear while the dose response function for double‐strand breaks is curved, indicating that it is the sum of both linear and quadratic components. We discuss factors that determine the sensitivity of the method and indicate approaches to make possible the quantitation of strand breaks in the DNA of cells irradiated with sublethal doses.
Direct N‐terminal sequence analysis of rat liver plasma membrane glycoproteins separated by two‐dimensional polyacrylamide gel electrophoresis
Authors: Benjamin Timothy , Niu Chien‐Hua , Parmelee David C. , Huggett Anthony C. , Yu Betty , Roller Peter P. , Thorgeirsson Snorri S.
Published: May 2005

Nine previously uncharacterized membrane glycoproteins from normal rat liver have been analyzed by amino acid sequencing from two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) after transblotting to Immobilon‐P membranes. Three of these components show altered levels of expression in liver tumors. A single electroblotted polyacrylamide gel yielded sufficient quantities of these glycoproteins for amino acid sequencing and the N‐terminal structure could be determined for four of them. The remaining five glycoproteins of interest were not sequenceable in this manner, presumably because they had blocked N‐termini. Prior to electrophresis, two enrichment methods were applied to the crude liver membrane preparations: affinity chromatography with concanavalin A to isolate the plasma membrane glycoproteins and then fast protein liquid chromatography on Superose 12 to obtain components having a specific range of molecular weights. These materials were next subjected to 2‐D PAGE using pH 4–6 carrier ampholytes in the first dimension and 7.5% sodium dodecyl sulfate gels in the second. The proteins were then electroblotted to Immobilon‐P membranes and located by staining with Coomassie Brilliant Blue R‐250. Our results demonstrate that N‐terminal sequencing (gas‐phase) can be achieved on polypeptides obtained from approximately 250 μg of total glycoproteins applied to a single 2‐D gel.
Composition and genetic variability of proteins from nuclear fractions of mouse (DBA/2J and C57BL/6J) liver and brain
Authors: Jungblut Peter , Zimny‐Arndt Ursula , Klose Joachim
Published: May 2005

Proteins from nuclear plasma of mouse liver and brain and from the nuclear membranes of mouse liver were separated by two‐dimensional electrophoresis. For the purpose of comparison, liver cytosol proteins were also investigated. The protein samples were prepared from two inbred strains of the mouse (DBA/2J, C57BL/6J) and their hybrids. The patterns obtained were compared with regard to the composition and genetic variability (qualitative and quantitative variants) of proteins from different nuclear fractions and organs. The percentage (> 30 %) of spots common to different organs (liver, brain), but from the same nuclear fraction (plasma) was greater than the percentage (< 20 %) of spots common to different cell and nuclear fractions (cytosol, nuclear plasma and nuclear membranes) of the same organ (liver). Quantitative genetic variants occurred much more frequently than qualitative genetic variants (5.1 % vs. 0.2 %; liver nuclear plasma). The incidence of genetic variants was much higher in liver (5.3 %) than in brains (0.0 %), and higher in solubilized nuclear proteins (5.3 %) than in structure‐bound nuclear proteins (2.1 %).
Human alpha‐1‐antitrypsin subtyping by hybrid isoelectric focusing in miniaturized polyacrylamide gel
Authors: Alonso Antonio
Published: May 2005

The polymorphism of alpha‐1‐antitrypsin (PI) has been studied by hybrid isoelectric focusing in miniaturized immobilized pH gradient gels, with an interelectrode distance of 55 mm, in two narrow ranges of pH 4.35–4.75 and 4.35–4.55, following rehydration with pH 4.2–4.9 carrier ampholytes. The use of the separator N‐(2‐acetamido)‐2‐aminoethanesulfonic acid (ACES) in combination with carrier ampholytes for gel rehydration has been shown to enhance PI band sharpness. The influence of different additives (sucrose, sorbitol and glycerol) on the PI band pattern has also been evaluated. Glycerol has been shown to be responsible for the change in the relative mobility of the M3 band. The analysis of the minor M‐7 isoprotein zone by hybrid isoelectric focusing followed by silver staining has permitted a more reliable classification of PIM subtypes. A population study carried out with 164 unrelated individuals living in Spain is also presented.
Microheterogeneity of orosomucoid in pathological conditions
Authors: Hansen John‐Erik S. , Bøg‐Hansen Thorkild C. , Pedersen Bo , Neland Keld
Published: May 2005

Studies of orosomucoid (α1‐acid glycoprotein) in human serum have revealed that orosomucoid is a mixture of molecules with differences in the glycan chains. This microheterogeneity has been studied using crossed affinoimmuno‐electrophoresis with the lectin concanavalin A which binds to biantennary glycans. The relative proportions of the three orosomucoid subtypes are altered in various pathological conditions independently of the total serum orosomucoid concentration. There are reproducible differences in microheterogeneity patterns between some pathological conditions: Acute tissue injury or inflammation results in a high proportion of orosomucoid with biantennary glycans. Conditions with increased estrogen levels are associated with a high proportion of orosomucoid with tri‐ or tetraantennary glycans and a low total serum orosomucoid concentration. Chronic inflammation also seems to be associated with a high proportion of orosomucoid with tri‐ or tetraantennary glycans but with a high total serum concentration of orosomucoid. Other diseases, such as cancer, can not be associated with any specific microhetero‐geneity pattern. The microheterogeneity pattern in these conditions seems to be determined by disease activity and unspecific inflammation in surrounding tissues.
Lectin affinity electrophoresis of alkaline phosphatase for the differentiation of bone and hepatobiliary disease
Authors: Rosalki Sidney B. , Foo A. Ying
Published: May 2005

An affinity electrophoresis procedure is described for the separation and quantification of the bone‐ and liver‐derived fractions of alkaline phosphatase in plasma. Separation is carried out on cellulose acetate membrane pre‐soaked with buffer containing wheat germ lectin. The electrophoretic mobility of the bone enzyme is preferentially retarded by the lectin and this fraction is well separated from the liver fraction. After separation, enzyme activity is demonstrated by staining using an indigogenic alkaline phosphatase substrate incorporated in agar gel, and the stained fractions quantified by densitometry. The procedure has low imprecision, good linearity, and the activities of the bone and liver fractions correlate well with values obtained using nonelectrophoretic quantification methods. The procedure is especially suitable for use in the diagnostic laboratory.
Electrophoretic behavior and partial characterization of disease‐associated serum forms of gammaglutamyltransferase
Authors: Sacchetti Lucia , Castaldo Giuseppe , Salvatore Francesco
Published: May 2005

We have recently devised an improved procedure for the rapid electrophoretic separation of multiple forms of serum gamma‐glutamyltransferase (GGT). This procedure is based on the separation on cellulose acetate strips, usually employed for lipoprotein electrophoresis, followed by visualization with a fluorescent reagent. The method is highly sensitive and the fractions are more clearly resolved than with other, procedures. Reference intervals have been evaluated in the sera from 142 healthy subjects and the patterns (two GGT forms comigrating with alpha1 and alpha2‐globulin) are reproducible. In 150 sera from patients with various hepatobiliary diseases (including neoplasias), acute pancreatitis and non liver‐involving neoplasias, we observed some disease‐specific GGT forms: an albumin comigrating enzyme (Alb‐GGT) specific of liver neoplasia; a gamma‐globulin comigrating GGT (gamma‐GGT) and a nonmigrating isoform (dep‐GGT) both specifically associated to extrahepatic jaundice. Multiple lipoprotein fraction precipitation showed that beta‐gamma‐ and dep‐GGT are complexes between GGT and low density lipoprotein and very low density lipoproteins (LDL + VLDL), and that some of the alpha1‐GGT from cirrhotic patients is a complex between GGT and high density lipoprotein (HDL). GGT fractions from normal subjects and Alb‐GGT from patients with liver neoplasia do not appear to be complexed with lipoproteins. The amount of GGT complexed with LDL + VLDL differs in various hepatobiliary diseases, and using a cutoff of 20 U/L it is possible to use this test to distinguish non‐cholestatic diseases from liver malignancies (i. e. in monitoring cirrhotics and other high risk groups of patients as to the possible evolution to liver cancer, in patients with gastrointestinal tumor which frequently metastasizes to the liver). Furthermore, treatment of GGT with neuraminidase confirms the association of sialic acid with all the GGT fractions, although Alb‐GGT is more sensitive to this treatment than is beta‐GGT. Our study, indicating the existence of clinical biochemistry correlations between isoenzyme patterns of serum GGT and diseases of hepatobiliary nature including neoplasias, opens up the possibility of enlarging the vocabulary of biochemical signals in these disorders. Furthermore, the lipoprotein‐precipitated fractions of serum GGT are an additional test for discriminating hepatocarcinoma from noncholestatic chronic hepatitis and cirrhosis.
Simultaneous phenotyping of erythrocyte acid phosphatase and esterase D by nonequilibrium agarose isoelectric focusing
Authors: Dimo‐Simonin Noëlle , Brandt‐Casadevall Conxita , Gujer Hans‐Rudolf
Published: May 2005

Nonequilibrium agarose isoelectric focusing in a pH 4.5–7 gradient with 3‐(N‐morpholinopropanesulfonic) acid (MOPS) and taurine as chemical separators presents a fast, easy and reliable method for the simultaneous determination of the common erythrocyte acid phosphatase and esterase D phenotypes in hemolysates and dried bloodstains. The application of the method to experimental and casework bloodstains is described.
Detection of human serum amyloid A protein in very low density – and high density lipoproteins of patients after acute myocardial infarction
Authors: Feussner Giso , Ziegler Reinhard
Published: May 2005

Two acute‐phase proteins have been identified in very low density (VLDL)‐ and high density lipoproteins (HDL) of patients after acute myocardial infarction. Both proteins have a relative molecular weight of 11 000 and isoelectric points pI 6.08 and 6.27, and do not contain cysteine or sugar residues. Polyclonal antibodies to these acute phase reactants did not cross‐react with other serum apolipoproteins. Evidence is given that both proteins are polymorphic forms of the human serum amyloid A protein.
Essential problems in quantification of proteins following colloidal staining with Coomassie Brilliant Blue dyes in polyacrylamide gels, and their solution
Authors: Neuhoff Volker , Stamm Reinhard , Pardowitz Iancu , Arold Norbert , Ehrhardt Wolfgang , Taube Dieter
Published: May 2005

Quantitative determination of stained proteins following polyacrylamide gel electrophoresis (PAGE) is of increasing interest especially since computer‐aided densitometers have become available as well as recipes for sensitive and background‐free staining with Coomassie Brilliant Blue dyes. However, avoidance of separation artifacts is not the only essential prerequisite for quantitative evaluation. The local particle density of a protein in a given gel is of critical importance since it determines its stainability. Depending on local protein concentration, the dye binding to the same amount of a given protein differs considerably. Since the stainability of proteins using colloidal staining procedures, as with Coomassie Brilliant Blue dyes, is time‐dependent and, in addition, also dependent on the pore size of a given polyacrylamide gel used for PAGE, calibration curves for quantitative determinations have to be prepared in polyacrylamide gels of the same composition as used for PAGE. Staining conditions also have to be identical for calibration gels and gels under analysis. If, however, a set of calibration curves is prepared for different staining times, it is possible to calculate a generalized calibration curve, allowing for quantitative evaluation with flexible staining time. Furthermore, and in consequence of the implications due to particle density, quantitative determination via densitometry is only possible by determining the protein amount of each single measuring point (pixel) via its absorbance on the basis of a calibration curve. Since the particle density is inherent in a calibration curve, the final summation of the protein amount per pixel will give values close to reality.
Electrophoresis at elevated hydrostatic pressure of the multiheme hydroxylamine oxidoreductase
Authors: Masson Patrick , Arciero David M. , Hooper Alan B. , Balny Claude
Published: May 2005

The behavior of the multiheme protein hydroxylamine oxidoreductase (HAO) in polacrylamide gel electrophoresis was studied at hydrostatic pressures up to 3 kbar at 25°C. Due to the limited working volume of the high pressure vessel, the electrophoresis cells were miniaturized. A microcell which accommodates 6 capillary gel tubes is described. Between 1 bar and 1.5 kbar the enzyme did not undergo structural changes detectable in the gel system. At approximately 2 kbar the active form of the enzyme was partially dissociated. At higher pressures, the enzyme was converted to forms which were irreversibly inactive and had a higher apparent molecular mass, suggesting aggregation or denaturation.
Micro two‐dimensional gel electrophoresis of serum and testis esterases from different strains of mice
Authors: Abou‐Haīla Aīda , Fain‐Maurel Marcelle‐Anne
Published: May 2005

Two‐dimensional electrophoresis with time‐dependent polyacrylamide gradient gel electrophoresis (PAGGE) in the second dimension was applied to the separation of native molecular forms of esterases from serum and testis of four strains of mice (C57BL/6J, Swiss OF1, F1 hybrid derived from these two populations and Tfm). In Phast System, a modified pH 3–9 gradient, a linear 8–25 % gel gradient and a migration time corresponding to 300 Vh, were found to provide the best conditions for esterase analysis. About 70 esterase‐active fractions could be separated with good reproducibility. The variants were characterized by their pI (3.9–7.35), their relative mobility and the visual estimation of their susceptibility towards neuraminidase and different esterase inhibitors. In the two tissues, the distribution of the esterase variants corresponded to a 50–500 kDa molecular mass range of calibration proteins, but most of the serum and testis‐specific isoforms were confined to the 59–72 kDa range. All serum variants contained a terminal N‐acetylneuraminic acid residue, whereas only the testicular esterases in common with those in serum appeared sensitive to neuraminidase. Cholinesterases with a low relative mobility and carboxylesterases with a high relative mobility were detected in serum, while carboxylesterases accounted for the greatest part in the testis which also contained cholinesterases and acetylesterases. Minor interspecies differences were found between C57BL/6J and Swiss OF1 esterases. The expression of two variants which differed between these two species seemed intermediate for the hybrid originating form these two populations. Two new spots were detected in the two‐dimensional map of esterases from the strain bearing the Tfm mutation.
Synergistic induction of polypeptides by tumor necrosis factor and interferon‐gamma in cells sensitive or resistant to tumor necrosis factor: Assessment by computer based analysis of two‐dimensional gels using the PDQUEST system
Authors: Beresini Maureen H. , Sugarman Barry J. , Shepard H. Michael , Epstein Lois B.
Published: May 2005

Tumor necrosis factor (TNF) synergistically enhanced the antiproliferative activity of interferon‐gamma (IFN‐γ) in both TNF‐sensitive and TNF‐resistant variants of the cervical carcinoma line, ME‐180. TNF alone had no apparent effect on the levels of synthesis of individual proteins in either of these variant cell lines as assessed by computerized two‐dimensional gel analysis of cell lysates using the PDQUEST system. However, IFN‐γ enhanced the levels of 18 polypeptides and suppressed the levels of 10 polypeptides in both cell lines. When used in combination in both cell lines, TNF and IFN‐γ induced the synthesis of 10 polypeptides that were not induced by either agent alone. These synergistically induced polypeptides may be crucial to the mechanism of the synergistic antiproliferative action of TNF and IFN‐γ in ME‐180 cells.
Steady state electrolysis and isoelectric focusing
Authors: Hagedorn Rolf , Fuhr Günter
Published: May 2005

The properties of pH gradients formed by stationary electrolysis of weak mobile or fixed electrolytes are analyzed. The model uses the appropriate balance equations and those of chemical equilibria. It is shown how the equation of the current density has to be modified for considering that fraction of current that is associated with the diffusion of neutral buffer molecules within a pH gradient. Furthermore it is shown that the pH gradients themselves give rise to water production within the gradient and that essential properties of the steady state are related to chemical reactions between the electrolyte constituents. The differential equations describing the gradients of the concentration of a given component, the pH, conductivity and potential are explicitly formulated in relation to those reactions. The equations are solved numerically and the significance of the results for isoelectric focusing is discussed. The experimental conditions to reach shallow and smooth pH gradients exhibiting sufficient ionic strength are formulated.
The use of charge‐coupled devices in the quantitative evaluation of images, on photographic film or membranes, obtained following electrophoretic separation of DNA fragments
Authors: Boniszewski Zbigniew A. M. , Comley John S. , Hughes Barry , Read Christopher A.
Published: May 2005

Charge‐coupled devices can provide an excellent means for the quantification of DNA band images on membranes and photographic film. However, proper consideration must be given to important parameters such as signal‐to‐noise ratio and resolution, especially in applications which demand the use of large format media. When employed in the direct imaging of chemiluminescent blots, the charge‐coupled device can provide equal or better sensitivity than that obtained by indirect methods using film, with the additional advantages of wide dynamic range and freedom from the vagaries of film processing.
A high resolution two‐dimensional gel electrophoresis and silver staining protocol demonstrated with nuclear matrix proteins
Authors: Cupo James F. , Lidgard Graham P. , Lichtman William F.
Published: May 2005

An improved two‐dimensional gel electrophoresis procedure has been developed utilizing isolated nuclear matrix proteins. The proteins of the cellular nuclear matrix are tissue specific. They are an example of a protein set whose two‐dimensional electrophoretic patterns afford much information of clinical significance. However, current two‐dimensional gel techniques were not completely satisfactory for the small amounts of protein present in tissue samples. There was a need for a two‐dimensional gel procedure which was capable of increased sensitivity and resolution and at the same time was reliable and reproducible. This has been accomplished by implementing several modifications to the current two‐dimensional gel procedures. In addition, changes were introduced in the silver staining process of the gels to increase the signal to background ratio. The overall procedure affects a dramatic increase in the resolution and clarity of the proteins visualized on two‐dimensional gels and is no more laborious than current techniques.
Combination of two‐dimensional gel electrophoresis with microsequencing and amino acid composition analysis: Improvement of speed and sensitivity in protein characterization
Authors: Eckerskorn Christoph , Lottspeich Friedrich
Published: May 2005

High‐resolution two‐dimensional polyacrylamide electrophoresis (2‐DE) is commonly used as an analytical approach to resolve and detect most of the numerous protein species of an organism. However, the isolation of microgram amounts of protein in a 2‐DE spot in a form suitable for microsequence analysis and amino acid composition analysis is a key step in the chemical characterization of these proteins. With the development of chemically inert membranes it is now possible to retain proteins present in low quantities from the polyacrylamide matrix with high yields. The immobilized proteins are suitable for direct sequence analysis and amino acid composition analysis. The combination of protein chemical and electrophoretic techniques makes it possible to obtain chemical information from subpicomole quantities of protein, resulting in the availability of a new set of biologically important proteins for structural analysis. This paper summarizes the methods and strategies for the chemical protein analysis of 2‐DE spots in our laboratory.
Experimental and theoretical dynamics of isoelectric focusing: IV. Cathodic, anodic and symmetrical drifts of the pH gradient
Authors: Mosher Richard A. , Thormann Wolfgang
Published: May 2005

The production of anodic, cathodic and symmetrical drifts of a pH 3.5–10 gradient formed by isoelectric focusing in polyacrylamide gels is demonstrated experimentally by manipulation of the electrolyte concentrations. Experimental behavior is reproduced by computer simulation of a model mixture of 15 hypothetical carrier ampholytes whose pIs span the pH range 3 – 10. The mechanism which produces the drifts is elucidated and approaches to minimize such drifts are discussed. The data suggest why most experimentally observed drifts are cathodic.
Capillary zone electrophoretic separation of cyclodextrins with indirect UV photometric detection
Authors: Nardi Annalisa , Fanali Salvatore , Foret František
Published: May 2005

A method for the separation of cyclodextrins by capillary zone electrophoresis with indirect detection is described. It is based on the formation of inclusion complexes with benzoate which simultaneously forms a UV absorbing constituent of the background electrolyte used for the separation.
Characterization of artifical liposomes with free‐flow electrophoresis
Authors: Kessler Reinhard , Manz Hans‐Jörg
Published: May 2005

Free flow electrophoresis was used to examine the influence of active substances, lipid composition and preparation method on the surface charge of the liposomes. It is also possible to test the homogeneity of a liposome population with FFE.
Comprehensive two‐dimensional gel protein databases offer a global approach to the analysis of human cells: The transformed amnion cells (AMA) master database and its link to genome DNA sequence data
Authors: Celis Julio E. , Gesser Borbala , Rasmussen Hanne Holm , Madsen Peder , Leffers Henrik , Dejgaard Kurt , Honore Bent , Olsen Eydfinnur , Ratz Gitte , Lauridsen Jette B. , Basse Bodil , Mouritzen Solveig , Hellerup Marianne , Andersen Annette , Walbum Else , Celis Ariana , Bauw Guy , Puype Magda , Damme Jozef Van , Vandekerckhove Joël
Published: May 2005

A total of 3430 polypeptides (2592 cellular; 838 secreted) from transformed human amnion cells (AMA) labeled with [35S]methionine were separated and recorded using computer‐aided two‐dimensional (2‐D) gel electrophoresis. A master 2‐D gel database of cellular protein information that includes both qualitative and quantitative annotations has been established. The protein numbers in this database differ from those reported in an earlier version (Celis et al. Leukemia 1988,2, 561–602) as a result of changes in the scanning hardware. The reported information includes: percentage of total radioactivity recovered from the gels (based on quantitations of polypeptides labeled with a mixture of 16 14C‐amino acids), protein name (including credit to investigators that aided identification), antibody against protein, cellular localization, (nuclear, 40S hnRNP, 20S snRNP U5, proteasomes, endoplasmic reticulum, mitochondria, Golgi, ribosomes, intermediate filaments, microfilaments and microtubules), levels in fetal human tissues, partial protein sequences (containing information on 48 human proteins microsequenced so far), cell cycle‐regulated proteins, proteins sensitive to interferons α, β, and γ, heat shock proteins, annexins and phosphorylated proteins. The results presented should be considered as the initial phase of a joint effort between our laboratories to undertake a general and systematic analysis of human proteins. Using this integrated approach it will be possible to identify phenotype‐specific proteins, to microsequence them and store the information in the database, to identify the corresponding genes, to search for homology with previously characterized proteins and to study the function of groups of proteins (pathways, organelles, etc.) that exhibit interesting regulatory properties. In particular, the 2‐D gel protein database may become increasingly important in view of the concerted effort to map and sequence the entire human genome.
The MRC‐5 human embryonal lung fibroblast two‐dimensional gel cellular protein database: Quantitative identification of polypeptides whose relative abundance differs between quiescent, proliferating and SV 40 transformed cells
Authors: Celis Julio E. , Dejgaard Kurt , Madsen Peder , Leffers Henrik , Gesser Borbala , Honore Bent , Rasmussen Hanne Holm , Olsen Eydfinnur , Lauridsen Jette B. , Ratz Gitte , Mouritzen Solveig , Basse Bodil , Hellerup Marianne , Celis Ariana , Puype Magda , Damme Josef Van , Vandekerckhove Joel
Published: May 2005

A new version of the MRC‐5 two‐dimensional gel cellular protein database (Celis et al., Electrophoresis 1989, 10, 76–115) is presented. Gels were scanned with a Molecular Dynamics laser scanner and processed by the PDQUEST IITM software. A total of 1895 [35S]methionine‐labeled cellular polypeptides (1323 with isoelectric focusing and 572 with nonequilibrium pH gradient electrophoresis) are recorded in this database, containing quantitative and qualitative data on the relative abundance of cellular proteins synthesized by quiescent, proliferating and SV40 transformed MRC‐5 fibroblasts. Of the 592 proteins quantitated so far, the levels of 138 were up‐or down‐ regulated (51 and 87, respectively) by two times or more in the transformed cells as compared to their normal proliferating counterparts, while only 14 behaved similarly in quiescent cells. Seven MRC‐5 SV40 proteins, including plastin and two interferon‐induced proteins, were not detected in the master MRC‐5 images. The identity of 36 of the transformation‐sensitive proteins whose levels are up or down regulated by two times or more was determined and additional information can be transferred from the master transformed human epithelial amnion cells (AMA) database (Celis et al., Electrophoresis 1990, 11, 989–1071) for those polypeptides of known and unknown identity that have been matched to AMA polypeptides. As more information is gathered in this and other laboratories, including data on oncogene proteins and transcription factors, this comprehensive database will outline an integrated picture of the expression levels and properties of the thousands of protein components of organelles, pathways and cytoskeletal systems that may be directly or indirectly involved in properties associated with the transformed state.
A convenient gel holder for preparative electrophoretic separation of aggregated bacterial proteins
Authors: Groschup Martin H. , Boschwitz Jeff , Timoney John F.
Published: May 2005

A simple custom‐made gel holder for preparative SDS‐PAGE to separate aggregated bacterial antigens is described. The gel holder fits easily into commercially available gel electrophoresis apparatus and proteins or peptides are collected in a stream of distilled water through a channel in the gel. The performance of the device was illustrated by the successful separation and purification of fragments of aggregated Streptococcus equi M protein and of Erysipelothrix rhusiopathiae protective antigens. Yields of up to 1.2 mg per run were obtained. The purified proteins retained immunological reactivity and were of sufficient purity for amino acid compositional and sequence analysis.
Information on DNA conformation derived from the Ferguson plot of DNA fragments of up to 9 kb in size, using polyacrylamide gel electrophoresis in a discontinuous buffer system
Authors: Orbán László , Chrambach Andreas
Published: May 2005

The Ferguson plot in polyacrylamide gel electrophoresis (PAGE) (15 %CDATD, moving boundary electrophoresis buffer system operative at pH 8.9, 4°C, 8 mA/cm2 of gel) of DNA fragments up to 9.4 kb in size was found to exhibit a linear segment at polyacrylamide concentrations starting at 3 %T and undergoing a gradual transition into a concave segment at higher gel concentrations, confirming previous findings by Stellwagen [1]. The larger the DNA, and the higher the gel concentration, the less extended the linear and the more extended the concave segment of the plot. The lowest %T of the linear range for DNA in polyacrylamide remains unknown since mobilities at nongelling concentrations below 3%T have not as yet been measured. As previously suggested [1, 2], the transition from the linear to the concave segment corresponds to that from the randomly oriented DNA to the anisotropically stretched “reptating” DNA. For a DNA of 9.4 kb in size, the end of the linear range of the Ferguson plot can be extended from 3.5 to 5 %T when 15 % DATD rather than 2.5 % Bis is used to crosslink the polyacrylamide. Increasing the temperature of PAGE from 4°C to 25 and 50°C widens the linear segment progressively, indicating an increasingly random orientation with rising temperature. When current density is increased from 8 to 40 mA/cm2, the concave curvature of the Ferguson plot of DNA 1 to 9.4 kb in size decreases, suggesting a transition from a “reptating” to a randomly distributed molecule, due to increased Joule heat. Increasing the ionic strength from 0.01 to 0.04 M similarly appears to lead to a relative linearization of the Ferguson plot, suggesting a similar effect of Joule heat. Prestaining of DNA with ethidium bromide results in a decrease of both its size and its free mobility. This effect is exacerbated as the size of DNA increases from 0.1 to 9.4 kb.
Computer‐assisted statistical analyses of enzyme and ribosomal DNA electrophoretic polymorphism in Yersinia
Authors: Picard Bertrand , Denise Jean‐Baptiste , Decoux Guy , Goullet Philippe
Published: May 2005

The intra‐ and inter‐species differentiation of 90 strains of Yersinia belonging to six species were studied independently by computer‐assisted statistical analysis of data from enzyme electrophoretic polymorphism and ribosomal DNA (rDNA) restriction fragment length polymorphism. Two corresondence analyses (CA) demonstrated the concordance between the bacterial classification obtained from enzymatic and genomic data. This concordance was reinforced by an algorithm of the correspondence established between the two dendrograms drawn from previous computations. Comparison of CA with similarity analysis (also called “numerical taxonomy”) indicated that the intra‐ and inter‐species differentiation obtained by the two methods are similar. The advantage of CA is that it gives a synthetic geometrical representation of the results (factorial planes), displaying both the main features of the clusters of strains (location and dispersion) and their essential character (i.e.: enzyme electrophoretic variant, rDNA fragment size).
Application of silver staining to the rapid typing of the polymorphism of HLA‐DQ alleles, by enzymatic amplification and allele‐specific restriction fragment length polymorphism
Authors: Ju Li‐Ya , Gu Xue‐Fan , Larger Etienne , Krishnamoorthy Rajagopal , Charron Dominique
Published: May 2005

A rapid and highly sensitive silver staining method, originally developed for the detection of proteins, was slightly modified to detect nucleic acids in polyacrylamide gels. The second exons of the histocompatibility antigen HLA‐DQA1 and DQB1 genes were selectively amplified from genomic DNA by the polymerase chain reaction (PCR). Digestion of the PCR products by endonucleases, followed by their size separation on polyacrylamide gels and visualization by silver staining, allowed us to define the HLA‐DQ alleles of the genomic DNA. The in ensity of staining of digested PCR‐amplified DNA is linear from at least 8 to 18 ng for fragments of lengths ranging from approximately 40 to 200 bp. Thus, silver staining in combination with PCR and allele‐specific restriction fragment length polymorphism provides a simple, safe, and rapid method for accurate definition of HLA‐DQ alleles at the nucleotide level in the clinical typing laboratory.