bluuebunny/crossref_metadata_2025_split huggingface dataset:
Alkali enhancement of protein staining on nitrocellulose
Authors: Sutherland Mark W. , Skerritt John H. 
Published: May 2005

AbstractBrief alkali treatment of nitocellulose‐bound protein markedly enhanced subsequent staining of protein with India ink or colloidal gold but not Amido Black. Following alkali treatment, India ink staining approaches colloidal gold staining in sensitivity and exceeds the latter method in clarity and economy. Furthermore, apparent protein‐to‐protein variation is reduced, allowing the method to be used for sensitive staining of protein transfers. Simple, high sensitivity staining of nitrocellulose‐bound protein using India ink can be achieved after alkali treatment in less than 2 hours.
Examination of isoelectric focusing and electrophoretic methods for resolving acidic proteins
Authors: Green Eric D. , Knecht David A. , Dimond Randall L. 
Published: May 2005

AbstractHighly acidic proteins are difficult to study using conventional electrofocusing and electrophoretic techniques. For example, the lysosomal enzymes from the cellular slime mold Dictyostelium discoideum have isoelectric points (pI) around 3–4 due to extensive phosphorylation and sulfation. To assess differences in anionic modifications among these lysosomal enzymes, we required methods that resolved acidic proteins on the basis of their net charge. The separation procedures needed to be rapid and convenient, allowing parallel analysis of a large number of samples. Using these criteria, we developed two methods, isoelectric focusing in horizontal agarose gels and native electrophoresis in vertical polyacrylamide gels, which are more useful than other available techniques. Since the mechanism by which each system separates proteins is different, both methods give important information about acidic proteins.
Epigenetic modification of murine Cu,Zn‐superoxide dismutase
Authors: Shibata Futoshi , Ogita Zen‐Ichi 
Published: May 2005

AbstractMurine Cu, Zn‐superoxide dismutase (SOD) from erythrocytes has three isozymes, SOD‐1A (pI 6.4), B (pI 5.7) and C (pI 5.0). It has been suggested that SOD‐1A is formed epigenetically by asymmetric modification of SOD‐1B where as SOD‐1C is produced by treatment of superoxide dismutase with hydrogen peroxide. On two‐dimensional gel electrophoretic analysis, each isozyme shows two forms, easily permuted into each other, and hence supposed to be in equilibrium. The zymogram patterns of murine liver, kidney, heart and brain are the same as those in erythrocytes although SOD activity levels are different in these tissues.
Isotachophoretic analysis of the pH dependent carrier properties of bovine serum albumin for rifamycin AF/013
Authors: Csordas Adam , Oefner Peter Josef , Bartsch Georg , Grunicke Hans , Lancini Giancarlo 
Published: May 2005

AbstractThe binding sites of bovine serum albumin for rifamycin AF/013 were examined by means of analytical capillary isotachophoresis. At pH 7.5–7.9, there was only one binding site on non‐defatted bovine serum albumin, while under the same conditions up to 20 binding sites could be detected on fatty acid‐free albumin. In both cases the interaction became nonspecific above pH 8.0, resulting in a large number of rifamycin AF/013 molecules bound to albumin. Co‐binding with oleic acid in vitro resulted in competition with all but the first high‐affinity binding site for rifamycin AF/013, corroborating the findings made with non‐defatted albumin. Considering albumin as a model, the results might explain the conflicting reports about the number of binding sites of DNA‐dependent RNA polymerases for rifamy cin AF/013. Within a sharp pH‐limit the highly specific binding becomes a nonspecific one. Moreover, the results suggest that the carrier function of bovine serum albumin might drastically change in vivo, depending on the pH of physiological fluids.
Subcellular localization of some wheat polypeptides revealed by two‐dimensional electrophoresis
Authors: Granier Fabienne , Thiellement Hervé , Ambard‐Bretteville Françoise , Rémy René 
Published: May 2005

AbstractCell fractionation experiments were undertaken in order to assign some of the spots revealed by two‐dimensional gel electrophoresis of seven‐day‐old wheat etiolated seedlings to plastids or mitochondria. Among the 766 spots of these two‐dimensional gels, 251 have been identified: 139 in etioplastic stroma, 139 in chloroplastic stroma, 59 in mitochondria and 23 in chloroplastic membranes. Among them, 83 are common to etioplastic and chloroplastic stroma fractions but only 11 spots are common to mitochondria and plastids sub‐fractions.
How to estimate the position of the mistranslated proteins on a two‐dimensional gel electrophoregram
Authors: Laughrea Michael , Filion Anne‐Marie , Boulet Louise 
Published: May 2005

AbstractA method for the estimation of amino acid misincorporation into ribosomal proteins is presented. The method is based on the observation that the horizontal mobility of the 52 E. coli ribosomal proteins is proportional to Q/N, where Q is the calculated charge of the protein and N designates the number of residues in the protein. Thus the mobility change caused by a change in charge, such as the replacement of arginine by cysteine, can be predicted and the gel fractions used for scintillation counting cut accordingly. The gel system used in this paper was not the O'Farrell system but a ribosomal system whose first dimension was run at pH 5 in the presence of 8 M urea.
Application of two dimensional electrophoresis to characterise hormonally sensitive proteins in the normal and abnormal uterus
Authors: Guest Julian F. , Elder Murdoch G. , White John O. 
Published: May 2005

AbstractProgesterone therapy of androgenized rats results in the elimination of histological abnormalities of the uterus associated with constant unopposed oestrogenic stimulation. Subcellular fractions prepared from uteri of normal, androgenized and progesterone treated animals have been analysed by two‐dimensional gel electrophoresis. In all fractions, protein changes were observed as a result of androgenization and subsequent progesterone therapy. The identity of some of these proteins has been assigned following peptide mapping. Following progesterone therapy, the relative proportion of cytosolic tubulins, tropomyosins and D‐glucose‐6‐phosphate dehydrogenase were increased. In the abnormal uterus, a protein with similarity to the B subunit of creatine kinase was present in the mitochondrial fraction but could not be detected in the cytosol. Subsequent to progesterone therapy, this protein was no longer detectable in the mitochondrial fraction whilst two molecular forms of the B subunit of creatine kinase appeared in the cytosol. These hormonally‐sensitive changes in both structural and enzymatic proteins, which are ubiquitous to most cell types, are discussed in relation to morphological differentiation of the uterus and the molecular mechanisms underlying cell growth.
Pollen allergen purification by three successive preparative electrophoretic steps
Authors: Demeulemester Claude , Peltre Gabriel , Panheleux Dominique , David Bernard 
Published: May 2005

AbstractWater soluble allergens of grass pollen (Dactylis glomerata) were purified by two successive steps of preparative isoelectric focusing in a flat bed of granulated gel and controlled by analytical isoelectric focusing in agarose gels. In order to separate components cofocusing in the same pH zone but having different electrophoretic mobilities, preparative zone electrophoresis in granulated gels was performed. The homogeneity of the fractions recovered was tested by isoelectric focusing and zone electrophoresis, followed by radioimmunodetection of the allergenic molecules on nitrocellulose prints. The major allergen of Dactylis glomerata pollen was purified with a yield of 7.5 mg from 10 g of pollen grains.
Degradation kinetics of an alkaline Immobiline in the frozen state
Authors: Astrua‐Testori Silvia , Pernelle Jean‐Jacques , Wahrmann Juan P. , Righetti Pier Giorgio 
Published: May 2005

AbstractThe degradation kinetics of Immobiline pK 9.3 (0.2 m in H2O) have been studied over a period of 2‐years in the frozen state. The apparent hydrolysis rate (derived from the shift of myosin spots in a two‐dimensional map) is 20% per year (or 10% if incorporation of acrylic acid, an hydrolysis product, is accounted for). Such reactions in frozen solutions are not uncommon (cf. the spontaneous mutarotation of glucose, Kiovsky, T. E. and Pincock, R. E., J. Amer. Chem. Soc. 1966, 88, 4704–4710).
Two‐dimensional analysis of membrane proteins with isoelectric focusing in immobilized pH gradients in the first dimension
Authors: Gianazza Elisabetta , Caccia Paolo , Quaglia Laura , Righetti Pier Giorgio , Rimpiläinen Marja A. , Forsén Raili J. 
Published: May 2005

AbstractTwo‐dimensional electrophoresis with isoelectric focusing in immobilized pH gradients in the first dimension was applied to the fractionation of hydrophobic proteins from the plasma membrane of a Streptococcus strain. The detergents incorporated into the first‐dimensional gel slab, especially the zwitterionic ones of the sulfobetaine series, interfere with protein transfer from the first to second dimension by formation of mixed micelles with sodium dodecyl sulfate molecules. In order to reduce their concentration an elution protocol was devised including: (i) fixation for 1 h in 50 % methanol ‐ 12 % acetic acid; (ii) washing with distilled water for 30 min; (iii) equilibration in concentrated Tris buffer; (iv) denaturation in 5 % sodium dodecyl sulfate ‐ 2 % 2‐mercaptoethanol. When counting the number of resolved spots in the protein patterns after two‐dimensional electrophoresis, the relative solubilizing efficiency of different detergents scored as follows: Nonidet P‐40 (NP‐40) > (3‐[3‐cholamidopropyl)dimethylammonio]‐1‐propananesulfonate (CHAPS) > N‐dodecyl‐N, N‐dimethylammonio‐3‐propanesulfonate (sulfobetaine SB 12).
Determination of relative amounts of silver stained proteins on two‐dimensional gels using internal calibrator
Authors: Stastny Jaroslav J. , Fosslien Egil 
Published: May 2005

AbstractProteins separated by isoelectric focusing of 20 samples and polyacrylamide gel electrophoresis of 10 samples in the same batch were stained with silver and were scanned with a computerized laser beam densitometer to obtain integrations values. A linear relationship was found between densitometric integration values (staining intensities) of proteins and their loads up to 600 ng (actin, carbonic anhydrase) or 1000 ng (albumin, creatine kinase), respectively. The same amount (250 ng) of applied albumin, actin, carbonic anhydrase and creatine kinase resulted in differences in staining intensities of these proteins; staining intensities of albumin and creatine kinase were similar, however: both were 1.8 X higher than the staining intensity of carbonic anhydrase and 2.3 X higher than of actin. Six human aortic intima proteins (ML‐1, ML‐2, P27, −14, P27,−18, P27,−19.5, P110) were selected for quantitative studies of the relationship between loads of intimal extract and the integration values of these proteins. Linear responses were found for these proteins in loads from 5 μL to 20 μL of intimal extract, corresponding approximately from 15 μg to 60 μg of total proteins applied to the focusing gel. When carbonic anhydrase was included with each sample, as an internal absorbance calibrator, we could express integration values of other proteins in relative amount in ng of carbonic anhydrase and thus compensate for variation in fixation, developing and staining conditions from batch to batch.
The effect of narrow pH range ampholytes upon resolution and protein detection methods following two‐dimensional electrophoresis
Authors: Marshall Thomas , Williams Katherine M. 
Published: May 2005

AbstractAmpholine pH 4–6 and Pharmalyte pH 4.2–4.9, 4.5–5.4 or 5–6 have been used to improve the resolution of the simplified technique of high resolution two‐dimensional electrophoresis. All give excellent resolution but Pharmalyte interferes with protein detection methods to an extent which increases with the pH range of the mixture. The effect is characterised by a progressive increase in the intensity and area of background stain associated with the anode and cathode regions of the two‐dimensional gels. It is minimal following Serva Blue R (Coomassie Blue) staining but severe following silver staining.
Electrophoretic detection of the haptoglobin polymorphism at the DNA level
Authors: David Fabrice , Lavadera Sandra , Lucotte Gérard 
Published: May 2005

AbstractDirect gene analysis of the haptogobin (Hp) polymorphism was carried out by Southern blotting using an Hp cDNA probe. Two types of polymorphism were observed: the first, after EcoRI, HindIII and PstI digestion, is due to an intragenic duplication and corresponds to the Hp‐α protein polymorphism; the second type, due to point mutation, was represented by two additional restriction sites for EcoRI and PstI. The univocal correspondence between the first type fragment length variants and protein polymorphism of the duplication type permits the establishment of the genotype at the DNA level, and mutational variants of the second type add new markers; the method described here also permits detection of the real genotype of Hp0 alleles.
Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels
Authors: Blum Helmut , Beier Hildburg , Gross Hans J. 
Published: May 2005

AbstractA new modification of silver staining is presented which utilizes two chemical properties of thiosulfate: image enhancement by pretreatment of fixed gels, and formation of soluble silver complexes which prevents unspecific background staining during image development. This procedure provides high sensitivity for proteins, RNA and DNA in the nanogram range on a colorless, transparent background. The performance of this method is documented by staining one‐and two‐dimensional patterns of plant leaf proteins. Moreover, we achieved, for the first time, the detection of the non‐structural, tobacco mosaic virus‐specific 126 kDa protein directly in the one‐dimensional protein pattern of infected protoplasts by a staining procedure.
Genetic polymorphism within cellular proteins of human peripheral lymphocytes analyzed by two‐dimensional electrophoresis: A family and population study of individual variations
Authors: Waldinger Dorothea , Cleve Hartwig 
Published: May 2005

AbstractGenetic polymorphism of human lymphocyte proteins was studied by two‐dimensional electrophoresis in the members of 12 families and in additional 20 unrelated individuals from Southern Germany. Cell lysates of phytohemagglutinin‐stimulated, [3H‐]leucine labeled peripheral lymphocytes were subjected to two‐dimensional electrophoresis. Among the approximate 300 labeled proteins on two‐dimensional fluorograms we found six polymorphic proteins. Five proteins, named p23 (Mr 23 000), p30 (Mr 30 000), p40 (Mr 40 000), p60 (Mr 60 000) and p66 (Mr 66 000), existed as two charge variants, an acidic (a) and a basic (b) variant. Three different electrophoretic phenotypes, two homozygous (a and b) and one heterozygous (ab), could be observed for the proteins p23, p40 and p66. The proteins p30 and p60 occurred in our population sample only in two phenotypes, a common homozygous (a) and a rare heterozygous (ab) phenotype. Preliminary results are presented for the sixth polymorphic protein p75 (Mr 75 000), which occurred in at least three or even more different electrophoretic variants, resulting in six or more phenotypes. The genetic basis of the protein variations was confirmed by the quantitative gene‐dosage dependence, the identity of the protein patterns in three monozygotic twin pairs and the transmission of the variants as Mendelian traits within the 12 families examined. Gene frequencies of the rare alleles of the five proteins which occurred in two charge variants ranged between 0.012 and 0.477 in a sample of 44 unrelated individuals from Southern Germany. Thus, the term genetic polymorphism is justified.
High resolution two‐dimensional polyacrylamide gel electrophoresis of basic myeloid polypeptides: Use of immobilized pH gradients in the first dimension
Authors: Strahler John R. , Hanash Samir M. , Somerlot Luke , Weser Johann , Postel Wilhelm , Görg Angelika 
Published: May 2005

AbstractA two‐dimensional polyacrylamide gel electrophoresis approach for the separation of urea/detergent solubilized basic proteins is presented. An immobilized pH gradient (IPG) gel, pH range 7–10, containing 8 M urea, 2% Nonidet P‐40 and 10 mM dithioerythritol was used for the first‐dimensional isoelectric focusing separation. Approximately 400 to 500 basic polypeptides from myeloblasts or neutrophils were visualized with silver staining. The IPG gel in some cases included carrier ampholytes in the same pH range. The carrier ampholyte‐modified (“hybrid”) IPG gel exhibited improved resolution of some polypeptides, but at the expense of reproducibility in the focusing of other polypeptides. Transfer of sample into the IPG gel was facilitated by a combination of low initial voltage, presence of carrier ampholytes in the sample and application of the sample to the surface of the gel within a containment collar. 6 M urea was required in the SDS equilibration solution for the efficient transfer and high resolution separation of polypeptides in the second‐dimensional sodium dodecyl sulfate‐gel. Absolute spot position was highly reproducible among gels prepared simultaneously. Standard deviations of ± 0.84 mm for the focusing and ± 1.25 mm for the molecular weight dimensions of 70 randomly selected spots were obtained. Integrated spot intensities ranged from 0.17 to 80 O.D. × mm2 with a median of 1.5 O.D. × mm2.
Improved separation of the common transferrin variants in gels containing pH 5–7 Ampholines and HEPES
Authors: Budowle Bruce 
Published: May 2005

AbstractAn ultrathin‐layer polyacrylamide gel isoelectric focusing technique is described for obtaining highly resolved transferrin (Tf) subtypes. N‐2‐hydroxyethylpiperazine‐N′‐2‐ethanesulfonic acid (HEPES) buffer was used to flatten the pH gradient in gels containing pH 5–7 Ampholines. The result was increased separation distances for the TfC subtype bands. The method is effective even with the most recent lots of pH 5–7 Ampholines which were not capable of providing an appropriate environment for effective TfC subtyping.
Studies by conventional and “low denaturing” isoelectric focusing on albumin microheterogeneity under normal conditions and in experimental nephrosis
Authors: Ghiggeri Gian M. , Candiano Giovanni , Queirolo Carlo , Gusmano Rosanna , Ginevri Fabrizio , Bertani Tullio , Remuzzi Giuseppe 
Published: May 2005

AbstractWith the aid of ultrathin‐layer isoelectric focusing, rat serum albumin was studied in a group of 6 normal rats and 4 rats affected by an experimentally induced nephrotic syndrome. In normal rats the albumin was microheterogeneous with pI values ranging from 4.7 to 6.1. In animals with the nephrotic syndrome the albumin was almost homogeneous, consisting only of the isoform with the lowest pI of 4.7. The patterns were altered by performing IEF in low denaturing media in presence of 0.5–2 Murea. Under these conditions two rat serum albumin bands with pI's close to 6.1 were observed. Urinary albumin was highly microheterogeneous in nephrotic rats (pI 4.7–6.1) and less heterogeneous in the normal group(pI4.7). Pooled urinary albumin from nephrotic rats was fractionated by preparative IEF in granulated gels in four isoforms (A1–A4) with decreasing pI. Both for serum and urinary albumin the fatty acid content in normal and nephrotic syndrome animals was higher in isoforms with low pI.
Increases in the 35kDa surfactant‐associated protein and its mRNA following in vivo dexamethasone treatment of fetal and neonatal rats
Authors: Phelps David S. , Church Susan , Kourembanas Stella , Taeusch H. William , Floros Joanna 
Published: May 2005

AbstractFetal and neonatal rats were treated with dexamethasone in vivo and the effect of this treatment on the 35kDa surfactant‐associated protein was examined. Dexamethasone treatment increased the levels of translatable mRNA for the 35kDa protein from day 19 of gestation until at least 6 days after birth. Prior to gestational day 19, no glucocorticoid effect was seen although the mRNA was detectable as early as gestational day 16. Similar results were seen when [35S]methionine‐labeled 35kDa protein was immunoprecipitated and subjected to two‐dimensional gel electrophoresis.
Characterization of subcellular particles by size, charge and apparent compressibility on the basis of mobility in agarose gel electrophoresis: Procedures of computer simulation
Authors: Tietz Dietmar , Gombocz Erich , Chrambach Andreas 
Published: May 2005

AbstractTwo procedures of computer simulation of the electrophoretic migration of a particle through agarose gel are described which allow for: (a) characterization of gel fiber dimensions as a function of gel concentration (gel standardization), (b) determination of particle radius and the dynamics of apparent particle compressibility during passage through the standardized gel, and (c) estimation of the net charge density of a particle by calculating its mobility at 0 % gel concentration. The common model underlying these simulations is based on the extended Ogston theory which probabilistically describes the migration of a particle through a random network of inert and non‐flexible fibers in terms of a “random space walk”. The first procedure, applicable to relatively rigid particles such as bacteriophages, standardizes the gel fiber on the basis of mobility values (cm/s)/(V/cm) at several gel concentrations of a single, or several, bacteriophages of known radius. Mobilities of an unknown bacteriophage are then used to simulate its physical properties. The second method, applicable to relatively non‐rigid particles such as plant viruses, uses 7 polystyrene particles of known radius to standardize the gel fiber, followed by simulation of virus properties on the basis of their mobilities at several gel concentrations. The techniques described are most appropriate for deriving physical properties of particles from their nonlinear plots of log (mobility) vs. gel concentration (Ferguson plots). They have the virtue of yielding the properties of native, hydrated gel fibers and particles.
Rehydratable ultrasensitive cellulose acetate substrate films for protease detection in ultrathin‐layer isoelectric focusing not interfering with subsequent silver staining of focused proteins in the gel
Authors: Kujat Richard 
Published: May 2005

AbstractA simple method of immobilizing protein on cellulose acetate by pretreating the films with methacryloxypropyltrimethoxysilane is presented. When a print is taken with such a substrate film the separating gel is not contaminated by the immobilized substrate, thus permitting location of focused proteins by means of silver stamina Dried films with immobilized substrate can be stored for several months before use Detection of alkaline and acid proteases is possible with a sensitivity of 0.3 ng/mm2.