bluuebunny/crossref_metadata_2025 huggingface dataset:
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

Ampholine 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

Direct 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

A 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

Genetic 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

A 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

An 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

With 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

Fetal 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

Two 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

A 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.
The purification and characterization of bovine salivary proteins by electrophoretic procedures
Authors: McLaren Robert D. , McIntosh James T. , Howe Gregory W.
Published: May 2005

Bovine salivary proteins have been shown to be correlated with bloat susceptibility in cattle. Due to the presence of the high molecular weight mucoprotein in whole saliva these proteins have been difficult to purify. This difficulty has now been overcome by using electrophoretic procedures. The purification protocol consisted of electrophoretic concentration of the saliva, separation of the proteins by polyacrylamide gel electrophoresis, excision of the protein bands followed by electroelution and concentration. Purified proteins were obtained from bands 4, 5, 6, 7, 8 and 9/10. The molecular weights of the intact proteins were between 40 300 and 112 400 on gel chromatography and the subunit molecular weights were between 15000 and 65 400. The isoelectric points were within the pH range of 3.6 to 5.1. Antisera were produced against protein bands 7 and 8 and these were used to determine antigenic relationships between proteins and as a means of quantifying the proteins by the Laurell rocket immunoassay.
Genetic studies of human apolipoproteins. II. A rapid one‐dimensional isoelectric focusing technique to characterize apolipoproteins A‐I, A‐II, A‐IV and C‐II of unfractionated plasma
Authors: Kamboh Mohammad I. , Ferrell Robert E. , Sepehrnia Bahman
Published: May 2005

A simple and rapid one‐dimensional analytical isoelectric focusing method followed by immunoblotting is described which detects genetic and biochemical variation in apolipoproteins A‐I, A‐II, A‐IV and C‐II without prior ultracentrifugation and delipidation of plasma samples. A polipoproteins separated on an isoelectric focusing gel are sequentially transferred to two nitrocellulose membranes and pairs of apolipoproteins are identified on each membrane by immunological reactions. The specificity of the primary antibody and the sensitivity gained by use of a second, alkaline phosphatase conjugated anti‐IgG allows the detection of free apolipoproteins in serum or plasma. A pH gradient 4–6.5 is suitable to identify the four apoproteins tested from a single gel and there is little loss of isoprotein resolution in sequential blots. Only 2 μL of whole plasma is required and results on 50 samples can be obtained from a single horizontal gel within one day.
Double replica electroblotting by oscillating electrotransfer
Authors: Johansson Karl‐Erik
Published: May 2005

Protein blotting has become an indispensable tool in biochemistry and in many blotting experiments it is desirable to obtain more than one replica from the gel, which would permit the use of different probes or staining of one membrane for protein and using the other(s) for probing. This has proved difficult. The different replicas, which can be produced by existing techniques, are far from identical. However, by using oscillating electrotransfer “double replica electroblotting”, two replicas can be obtained from one gel. The two replicas can be made almost identical by reversing the direction of the current in such a way that the efficient transfer time is increased with each period until the gel is almost completely depleted of proteins. The technique is useful for comparison of the reactions of different antisera with one or several antigen samples. Double replica electroblotting has been utilized for ordinary sodium dodecyl sulfate‐polyacrylamide gels, for two‐dimensional polyacrylamide gels and for thin gradient gels analyzed with the PhastSystem (Pharmacia). Double replica electroblotting can, in principle, be used with any blotting system where transfer from the gel to the new medium is achieved by electrophoresis, except in semi‐dry electroblotting with a discontinuous buffer system.
Visualization of molecular weight standards after electroblotting: Detection by means of corresponding antibodies
Authors: Bjerrum Ole J. , Hinnerfeldt Frank R.
Published: May 2005

The techniques available for detection of molecular weight standards after blotting are surveyed and evaluated. An identical immunodetection procedure for the antigens as well as for the molecular weight markers was evaluated by immunizing rabbits with the whole protein mixture of some commercially available calibration kits for sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Antibodies against both high and low molecular weight proteins were successfully raised albeit one rabbit failed to generate antibodies against ovalbumin. For immunodetection of rabbit myosin it was necessary to supplement the immunogen with bovine myosin. Best results were obtained by using more than one animal immunized with kits of different origin. Using alkaline phosphatase conjugated secconary antibodies, as little as 0.6 and 1.2 μg of the total low and high molecular weight mixture, respectively, are necessary for the immunoenzymatic detection of all bands. In fused rocket immunoelectrophoresis such antibodies are also useful for determination of the elution position of the molecular weight markers in gel filtration experiments.
Characterization of autoantigens and autoantibodies by immunoblotting
Authors: Elkon Keith B. , Bonfa Eloisa , Li‐Chu Jia , Parnassa Andrew P.
Published: May 2005

Immunoblotting has proven to be one of the most useful methods for detection of antibodies and partial characterization of proteins that are antigenic in the autoimmune diseases. The basic physicochemical characteristics (size and charge) of most of the major autoantigens have now been described. By combining various electrophoretic techniques with immunoblotting, a number of new applications have been reported. Two‐dimensional electrophoresis was used to fingerprint autoantigens and detect post‐translational modifications. Composite gel electrophoresis resolved ribonucleprotein complexes and was also used to analyze IgA, IgM autoantibodies and to determine the composition of cryoglobulins. Autoantibody heterogeneity was analyzed by isoelectric focusing. In some studies, the subclass, idiotype, or antigen binding specificity of autoantibodies could be determined by immunoblotting procedures. Coupled with improvements in antigen purity (synthetic peptides and recombinant proteins) and wider availability of monospecific antibodies, immunoblotting will continue to be a versatile tool in basic and clinical research in autoimmunity.
Factor B polymorphism in four Asian populations: Electrophoretic characterization of a subtype of “F” and of new variants in the Chinese
Authors: Suzuki Koichi , Charoenwongse Preeyachit , Lingao Augusto L. , Contractor Nafisa M. , Matsushita Sho , Sasazuki Takehiko , Matsumoto Hideo
Published: May 2005

The genetic polymorphism of factor B (BF) in four Asian populations was studied by immunofixation after agarose gel electrophoresis (AGE) in both veronal buffer (VB) and Tris/glycine/veronal buffer (TGVB). A suballele of the BF*F and three rare alleles were detected in a Chinese population. The subtype and the rare variants were further characterized by using isoelectric focusing in polyacrylamide gels (PAGIF) and by zymosan treatment of the serum samples. The subtype was identified as BFFb1, first defined in the Japanese and found to occur at a polymorphic frequency. One of the rare variants exhibited an intermediate mobility between BFF and BFS and seemed to be similar to BFF025. PAGIF, however, could clearly discriminate the two variants that had a similar mobility on AGE. The results obtained in this study suggest that conventional electrophoresis is not enough to characterize a new variant and to discriminate it from previously reported variants. The findings obtained on immunofixation after AGE/TGVB and in PAGIF must be added to those obtained by the standard method.
Electrophoretic variability in human hair: Comparative sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of body and head hair proteins
Authors: Gerhard Michael , Hermes Monika
Published: May 2005

The electrophoretic patterns of extracted proteins from human head hair were compared to those of proteins from other human hair species (pubic, breast, beard, arm, leg. axillary and eyebrow hair). The respective patterns (keratin phenotypes) were shown to be uniform throughout one individual's hair. Keratin typing could be performed successfully subsequent to ABH blood group determination by mixed cell agglutination with the same hair material.
Analysis by two‐dimensional gel electrophoresis of the polypeptide composition of pea mitochondria isolated from different tissues
Authors: Remy René , Ambard‐Bretteville Françoise , des Francs Catherine Colas
Published: May 2005

The polypeptide composition of purified mitochondria isolated from pea epicotyls was studied by one‐ and two‐dimensional electrophoresis. It was shown that two‐dimensional electrophoresis was necessary to clearly resolve polypeptides of molecuar weights ranging from 50 000 to 60 000. Mitochondria were fractionated into a soluble fraction corresponding to the matrix and an insoluble fraction containing membrane components. Spots characteristic of one or the other fraction were thus localized on the two‐dimensional gel map of whole mitochondria. In addition, a comparison was made of polypeptide composition in mitochondria isolated from etiolated and green leaves. It was shown that mitochondria from photosynthetic tissue differ from those on non‐photosynthetic tissue by quantitative and qualitative changes of their polypeptide composition. The most characteristic changes in green leaves are the loss of one polypeptide (Mr 35 000 and pI 6.3) and the quantitative increase of four other spots. These spots belong to the matrix compartment. Their apparent molecular weights are 97 000, 51 000, 43 000 and 15 000. Conversion of glycine to serine during the photorespiration process being characteristic of mitochondria from photosynthesizing tissue, we suggest that these spots could correspond to subunits of the glycine decarboxylase multienzyme complex.
Improved Nitro Blue Tetrazolium staining of creatine kinase isoenzymes following agarose gel electrophoresis or isoelectric focusing in polyacrylamide gels
Authors: Williams John , Marshall Thomas
Published: May 2005

An improved method is described for Nitro Blue Tetrazolium staining of creatine kinase isoenzymes following agarose gel electrophoresis or isoelectric focusing in polyacrylamide gels. The method allows the standard although transient fluorescent stain to be followed by Nitro Blue Tetrazolium staining to give a more permanent positive stain without background interference. It employs. 0.05 M para‐chloromercuribenzoate to block the adverse effects of sulfhydryl compounds (included in commercial creatine kinase‐reagents) upon Nitro Blue Tetrazolium staining.
Alpha‐1 acid glycoprotein analysis on immobilized pH gradients
Authors: Gianazza Elisabetta , Catto Nadia , Righetti Pier Giorgio , Bianchi‐Bosisio Adriana
Published: May 2005

By taking advantage of the insensitivity of immobilized pH gradients to high protein and salt loads, microheterogeneity and polymorphism of α1‐acid glycoprotein could be demonstrated on whole serum by conventional Coomassie Brilliant Blue G‐250 staining. Different running conditions were compared for optimal resolution.
Immobilized pH gradients: Effect of salts, added carrier ampholytes and voltage gradients on protein patterns
Authors: Righetti Pier Giorgio , Chiari Marcella , Gelfi Cecilia
Published: May 2005

Salts formed from strong acids and bases (e. g. NaCl, Na2SO4, Na2HPO4), presentin a protein sample applied to an immobilized pH gradient (IPG) gel, induce protein modification (oxidation of iron moiety in hemoglobin) already at low levels (5 mM) and irreversible denaturation (precipitation) at higher levels (>50 mM). This effect is due to production of strongly alkaline cationic and strongly acidic anionic boundaries formed by the splitting of the salt's ion constituents, as the protein zone is not and can not be buffered by the surrounding gel until it physically migrates into the gel matrix. Substitution of “strong” salts in the sample zone with salts formed by weak acids and bases, e. g., Tris‐acetate, Tris‐glycinate, Good's buffers such as (N‐[2‐aceta‐mido]‐2‐iminodiacetic acid (ADA)), (2‐[(2‐amino‐2‐oxoethyl)‐amino] ethanesulfonic acid (ACES)), (3‐[N‐morpholino] propane sulfonic acid (MOPS)), essentially abolishes both phenomena, oxidation and irreversible denaturation. Suppression of “strong” salt's effects is also achieved by adding, to the sample zone, carrier ampholytes in amounts proportional to the salt present (e. g. by maintaining a salt: carrier ampholytes molar ratio of at least 1:1). This suppression is due to the strong buffering power of the added carrier ampholytes, able to counteract drastic pH changes in the two moving boundaries. A reduction of these deleterious effects of strong salts is also achieved when the IPG run is performed at low voltage for a prolonged time (4 h at 500 V instead of only 1 h at 500 V, before switching to high‐voltage settings). Guidelines are given for trouble‐free IPG operations.
Automatic classification of two‐dimensional gel electrophoresis pictures by heuristic clustering analysis: A step toward machine learning
Authors: Appel Ron , Hochstrasser Denis , Roch Christian , Funk Matthieu , Muller Alex F. , Pellegrini Christian
Published: May 2005

The interpretation of two‐dimensional gel electrophoresis (2‐DGE) profiles can be facilitated by artificial intelligence and machine learning programs. We have incorporated into our 2‐DGE computer analysis system (termed MELANIE‐Medical Electrophoresis Analysis Interactive Expert system) a program which automatically classifies 2‐DGE patterns using heuristic clustering analysis. This program is a step toward machine learning. In this publication, we describe the classification method and the preliminary results obtained with liver biopsy electrophoretograms. Heuristic clustering is also compared to other classification techniques.
High resolution two‐dimensional electrophoresis and sodium dodecyl sulphate‐polyacrylamide gel electrophoresis of cheese proteins after rapid solubilisation
Authors: Marshall Thomas , Williams Katherine M.
Published: May 2005

The proteins of cheese are rapidly solubilised by heating to 95 °C in buffered 2 % sodium dodecyl sulfate, 5 % 2‐mercaptoethanol. Electrophoretic analysis of the solubilised proteins by either one‐dimensional sodium dodecyl sulphate‐polyacrylamide gel electrophoresis or high resolution two‐dimensional electrophoresis yields reproducible patterns characteristic of an individual cheese and its extent of ripening. The patterns reveal (i) the residual amounts of milk casein and whey proteins, and (ii) the appearance of casein degradation products, including pink‐violet components as detected by Coomassie Blue staining.
Demonstration and elimination of artifacts associated with gold staining of proteins on nitrocellulose membranes after separation by sodium dodecyl sulphatepolyacrylamide gel electrophoresis
Authors: Bath M. L.
Published: May 2005

Artifacts associated with gold staining of polypeptides separated by sodium dodecyl sulphate – polyacrylamide gel electrophoresis and transferred to nitrocellulose are eliminated by alkylation of reduced samples or by deletion of the reducing agent.
PI Mtoyoura: A new PI M subtype found by separator and hybrid isoelectric focusing
Authors: Yuasa Isao , Suenaga Kazuyuki , Umetsu Kazuo , Inagaki Osamu , Ikebuchi Jun , Ito Keiichi , Okada Kichiro
Published: May 2005

In the course of a population study of α1‐antitrypsin polymorphism by separator isoelectric focusing, a variant phenotype having a somewhat narrower spacing than PI M1M3 was observed in a Japanese blood donor. Family studies by hybrid isoelectric focusing in a carrier ampholyte‐supplemented immobilized pH gradient from 4.35–4.65 revealed that the products of the responsible gene, PI*Mtoyoura, were extremely close but slightly cathodal to those of PI*M1. The difference in isoelectric point between them corresponded to the resolving limit of isoelectric focusing. For this reason, although the propositus' father was deduced to have the genotype PI*M1/PI*Mtoyoura, the products of these two genes failed to form a double band pattern. Thus, the gene frequency for PI*Mtoyoura was unknown. These findings, however, indicate that a further microheterogeneity in the PI M subtype exists at least in the Japanese.
Somatic mutation studies in human lymphoid cells: The detectability of quantitative genetic variants in two‐dimensional gels
Authors: Kuick Rork D. , Hanash Samir M. , Strahler John R.
Published: May 2005

The feasibility of detecting quantitative genetic variants based on a decrease in the integrated intensity of polypeptide spots in two‐dimensional polyacrylamide gels of human lymphoblastoid cell clones was investigated. A battery of 65 spots on 115 gels was studied to determine the distribution of quantitative measures for spots where no mutation had occurred. The corresponding distribution for spots which have decreased integrated intensity as a result of a mutation at one of two alleles coding for the spot was investigated by quantitating spots for which mutations were known to have occurred. These two distributions allowed the estimation of the rates of false positive and false negative errors for any particular strategy aimed at detecting null mutations, and thus provides a basis for the design of efficient strategies. Our silver stained gels have sufficient reproducibility of spot integrated intensities so that, for situations in which the mutation rate is relatively high, it is practical to monitor a sub‐set of spots for null variants using the same digitized images as are used to detect structural variants.
Isoelectric focusing of group specific component in semen and vaginal stains on ultrathin immobilized pH gradient gels
Authors: Pflug Werner , Eberspächer Brigitte , Bäßler Gerhard
Published: May 2005

Subtyping of the group specific component in secretions of semen and vaginal fluids is impossible with conventional detection systems. By means of a highly sensitive alkaline‐phosphatase secondary antibody system the group specific component can reliably be‐detected in semen stains. Results with vaginal swabs were inconsistent and in saliva stains Gc activity could not be detected.
Heterogeneity in copper and glycan content of ceruloplasmin in human serum differs in health and disease
Authors: Hansen John‐Erik S. , Heegaard Peter M. H. , Jensen Søren P. , Nørgaard‐Pedersen Bent , Bøg‐Hansen Thorkild C.
Published: May 2005

Crossed immunoelectrophoresis of human serum revealed two heterogeneity types of ceruloplasmin with different electrophoretic migration. The two types both consisted of peptides with Mr 150 000, 100 000 and 45 000, which were interpreted as native ceruloplasmin and two hydrolytic fragments. The two types were different in copper content, and one type could reversibly be changed into the other. The glycan microheterogeneity of ceruloplasmin was analyzed by crossed affinommunoelectrophoresis with free Lens culinaris agglutinin (LCA) and wheat germ agglutinin (WGA). A third of the ceruloplasmin molecules, both high and low copper type, bound to LCA and two thirds to WGA. The heterogeneity and the microheterogeneity of ceruloplasmin in two groups of patient sera were compared to sera from healthy individuals. The ceruloplasmin type with respect to copper content was a much better factor than either glycan microheterogeneity or total serum concentration in discriminating between the three groups.
A double‐label two‐dimensional procedure for the analysis of membrane proteins
Authors: Wheeler Thomas T. , Jordan T. William , Ford Henry C.
Published: May 2005

A previously described double‐label two‐dimensional electrophoresis procedure (Wheeler et al., Anal. Biochem. 1986 159, 1–7) for the analysis of differences between two complex mixtures of soluble proteins has been modified to allow analysis of proteins requiring detergent for aqueous solubility. The samples are first disrupted by sonication and the insoluble proteins concentrated by high‐speed centrifugation. The proteins are then solubilized with sodium dodecyl sulfate and further concentrated in a centrifugal concentrator to achieve protein mixtures suitable for labeling with 14C and 3H by reductive methylation and subsequent two‐dimensional electrophoresis. The sample concentration step is quick, minimizes the concentration of sodium dodecyl sulfate in the final sample, and avoids the potential difficulties associated with lyophilization or precipitation. The modified procedure was applied to the analysis of erythrocyte membranes, platelets and isolated placental microvilli. The high resolving power of two‐dimensional gel electrophoresis is retained and the procedure is sensitive because the conditions of labeling allow substantial incorporation of radioactivity into protein despite the presence of detergent.
Comparative study of electrophoretic patterns of latex proteins from clones of Hevea brasiliensis
Authors: Arreguín Barbarín , Lara Paulina , Rodríguez Roberto
Published: May 2005

Latex serum proteins from Hevea brasiliensis were studied by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Proteins from whole serum and fractions isolated by gel chromatography on Ultrogel AcA 44 were analyzed. No qualitative clonal differences were found in the protein patterns of whole latex or in the fractions but laser densitometry revealed reliable quantitative differences in protein composition. Reproducible mobilities and molecular weights of selected bands were obtained both within single gels as well as in different gels, analyzing severallots of latex received at various times from a Hevea experimental field station. The clones compared were IAN 710, GV 31, GV 42; the first one had the highest rubber yields.
Optimized oligonucleotide probes for DNA fingerprinting
Authors: Schäfer Renate , Zischler Hans , Birsner Uli , Becker Andrea , Epplen Jörg T.
Published: May 2005

The three different simple repetitive oligonucleotide probes (CT)8, (CAC)5 and (TCC)5 were hybridized to a panel of human DNAs which had been digested with the restriction endonucleases Alu I, Hinf I and Mbo I. The resulting DNA fingerprints were analyzed and different parameters calculated, such as the maximal mean allele frequency and the average number of polymorphic bands per individual. The highest number of bands was obtained after hybridization of Hinf I digested DNA with (CAC)5. The probability of finding the same band pattern as in individual A in individual B is 2 × 10−8. The DNAs of monozygous twins show indistinguishable banding patterns and the bands are inherited according to the Mendelian laws. Thus this procedure reveals informative fingerprints that can be used for individual identification, e. g. in paternity testing and in forensic applications. In most of these experiments 32P‐labelled probes were employed, yet the biotinylated oligonucleotide (GACA)4 produced results which were equivalent to those obtained by hybridization with the 32P‐labelled probe (GACA)4.
Subtyping of orosomucoid 1 (ORM1) by isoelectric focusing in agarose and polyacrylamide gels
Authors: Thymann Mariann , Weidinger Sebastian
Published: May 2005

Genetic polymorphism of orosomucoid (ORM) has been demonstrated in several populations and comprises two structural gene loci, ORM 1 and ORM2. In Caucasians three common ORM 1 alleles have been shown, while the ORM 2 locus is almost monomorphic. ORM 1 phenotyping by isoelectric focusing in agarose or polyacrylamide gel combined with either print immunofixation or enzyme‐linked immunoblotting is described, and population and family data from Denmark and Southern Germany are given. It is proposed to use a different alpha‐numerical nomenclature for the phenotypes of the ORM 1 and ORM 2 systems.
The genetic haptoglobin polymorphism: Relevance of paternity assessment
Authors: Patzelt Dieter , Geserick Gunther , Schröder Helga
Published: May 2005

A practical method for haptoglobin subtyping is described utilizing fast sample preparation by means of batch adsorption to DEAE‐cellulose and subsequent isoelectric focusing of reductively cleaved samples. The expanded haptoglobin polymorphism leads to an increase of the theoretical paternity exclusion rate to approximately 33 %. Hence, the system appears to be highly attractive for paternity assessment.
Analysis of the genetic polymorphism of coagulation factor XIIIB (FXIIIB) by isoelectric focusing
Authors: Leifheit Hans‐Jürgen , Cleve Hartwig
Published: May 2005

The genetic variants of the coagulation factor XIIIB (FXIIIB) were analyzed by isoelectric focusing, carried out in agarose gels and followed by immunofixation. The FXIIIB phenotypes were visualized by a combined staining procedure with Coomassie Brilliant Blue R‐250 and silver nitrate. Improved resolution was accomplished in polyacrylamide gels by hybrid isoelectric focusing in immobilized pH gradients supplemented with carrier ampholytes. We examined a total of 1.604 unrelated, healthy individuals from Southern Germany. The frequencies for the FXIIIB alleles were B*1 = 0.7581, B*2 = 0.0843, B*3 = 0.1568 and B*4 = 0.0019. The theoretical exclusion rate for disputed paternity is 22.35 %.
Two new esterase D(ESD) variants revealed by isoelectric focusing in agarose gel
Authors: Weidinger Sebastian , Henke Jürgen
Published: May 2005

Using isoelectric focusing in thin‐layer agarose gel (AGIF) with the narrow pH range of 4.5–5.4, a high resolution of esterase D (ESD) isozyme banding patterns has been achieved. Some variant phenotypes which could not be distinguished from common ESD types by conventional electrophoresis have shown different patterns after AGIF. The IEF method permitted the distinction of two further variants in the ESD system, tentatively named ESD Rehren and ESD Ravensburg. We recommend, therefore, that for the classification of ESD phenotypes a high resolution IEF technique should be used.
The use of α2HS‐glycoprotein and group specific component in typing forensic blood samples for discriminative and investigative purposes
Authors: Westwood Sara A.
Published: May 2005

Methods are described for phenotyping α2HS‐glycoprotein (A2HS) and group specific component (GC) in plasma and blood‐stains. The methods have been developed to be sensitive, to provide unequivocal results and to give maximal information from minimal amounts of sample. In attempting to fulfill the last criterion, a new method is described for the simultaneous typing of α2 HS‐glycoprotein and group‐specific component from serum or plasma samples. The use of these proteins in determining the racial origin of a blood sample is also discussed.
The adsorption of large proteins in electrofocusing on immobilized pH gradients: II. Dependence on the oligomeric state of Immobiline
Authors: Fawcett John S. , Vicchio Domenick , Chrambach Andreas
Published: May 2005

Five proteins with molecular mass in excess of 200 kDa were found to adsorb onto gels during isoelectric focusing on immobilized pH gradients (IPGEF). To probe for the mechanism of that adsorption, the homogeneity of the six Immobiline preparations used to make IPGEF gels was tested. Five of these Immobiline preparations appear homogeneous in gel filtration of Sephadex G‐10. The sixth Immobiline (pK 9.3) exhibits a minor component eluting ahead of the major peak and comprising less than 4 % of the total Immobiline absorbing at 226 nm. The proportion of the minor component increases with column load. Major and minor components when isolated appear to equilibrate with one another. Judging by the results of mass spectrometry, all 6 preparations are free of small aggregates of less than 500‐600 Da molecular mass. Ultrafiltration of the Immobiline preparations through a membrane with 500 Da nominal cutoff leads to partial desorption of only 3 of the 5 adsorbed proteins. CHAPS is ineffectual in desorbing the 5 proteins from the IPG gel made with ultrafiltered Immobilines. None of the 6 Immobiline preparations used precipitates ferritin. All large proteins that adsorb onto IPGEF gels in the pH range 4–9.5 also adsorb onto commercial IPGEF gels in the pH range 4–7.
Two‐dimensional electrophoresis. Dual‐label autoradiographic analysis of human skin fibroblast and myoblast proteins by two‐dimensional polyacrylamide gel electrophoresis using immobilised pH gradients in the first dimension
Authors: Patel Ketan , Dunn Michael J. , Günther Siegfried , Postel Wilhelm , Görg Angelika
Published: May 2005

Horizontal two‐dimensional polyacrylamide gel electrophoresis with immobilised pH gradients in the first dimension has been applied to the analysis of human skin fibroblast and muscle myoblast total cell proteins. Excellent two‐dimensional separations of skin fibroblast proteins were obtained using pH 4–10 immobilised pH gradient gels with a long interelectrode distance (16 cm), but resolution was degraded, particularly of the more acidic proteins, by the use of shorter (10 cm) gels. Improved resolution of acidic and basic proteins was obtained using separate pH 4–7 and pH 7–10 immobilised pH gradient gels respectively in the first dimension. Two‐dimensional protein maps of skin fibroblast proteins were visualised both by silver staining and by autoradiography of samples labelled synthetically with [35S]methionine. Horizontal two‐dimensional electrophoresis, using pH 4–7 and pH 7–10 immobilised pH gradient gels in the first dimension, was applied to the analysis of protein samples from skin fibroblasts and muscle myoblasts dual‐labelled synthetically with [35S]methionine and [75Se]selenomethionine in an attempt to identify sets of proteins specific to each cell type. In addition, two‐dimensional maps or protein samples derived from normal individuals and patients with Duchenne muscular dystrophy were compared to search for protein changes associated with the disease state. Although sets of qualitative protein spot differences were observed by visual inspection of the two‐dimensional gels, more rigorous qualitative and quantitative analysis of the patterns using a computerised analysis system will be required to obtain the maximum amount of information from these data.
Preparation by two‐dimensional electrophoresis of proteins for antibody production: Antibodies against proteins whose synthesis is reduced by auxin in tobacco mesophyll protoplasts
Authors: Meyer Yves , Grosset Jean , Chartier Yvette , Cleyet‐Marel Jean‐Claude
Published: May 2005

Using phenol extraction from tobacco callus, we have prepared extracts with a high protein content. These proteins were separated in cylindrical non‐equilibrium pH gradient gels and visualized by dipping in sodium dodecyl sulfate (SDS)‐containing solution. Three gel sections, each containing proteins previously detected as abundantly synthesized in tobacco mesophyll protoplasts and whose synthesis is reduced by auxin application, were excised from each gel and collected. These proteins were further separated on slab SDS gels and protein bands were excised after Coomassie Brilliant Blue R‐250 staining and used to inject three rabbits. After one booster, highly specific antibodies were detected in their sera by ELISA and immunoblotting. Using these sera we have confirmed that the corresponding proteins are identical in callus and mesophyll protoplast and demonstrated that they are abundantly accumulated in tobacco roots but are undetectable in aerial organs and seeds.
Polypeptide patterns obtained by polyacrylamide gel electrophoresis of thylakoid membranes isolated from light and dark grown strains of Chlorogloea fritschii
Authors: Faulkner Michael
Published: May 2005

Sodium dodecyl sulphate‐polyacrylamide gel electrophoresis has been used to determine the difference between thylakoid polypeptide patterns of light and dark grown strains of the cyanobacterium Chlorogloea fritschii. There were only 2 prominent bands present in the dark grown strains, polypeptide Mr 50 000 and polypeptide Mr 90 000, also five fainter bands in the Mr range 45 000–66 200 corresponding to photosystem one, compared with the 32 bands present in the light grown strains. There was no obvious indication of the Mr 33 000 3‐(3,4‐chlorophenyl)‐1,1‐dimethyl‐urea binding protein. In addition the progressive daily development of the various photosystem components in the light and their relationship in photosynthesis was determined. It was observed that the increase of the relative concentrations of the photosystem two and phycocyanin components indicated their developments are mutually synchronized. The effect of light to dark and dark to light transfer on established strains was investigated. Appreciable loss of photosystem two components and the presence of an additional band Mr 22 500 of unknown function in the light to dark transfer, and little reactivation of the photosynthetic capabilities in the dark to light transfer was observed.
Report of workshop on cellular protein databases derived from two‐dimensional polyacrylamide gel electrophoresis
Authors: Neidhardt Frederick C.
Published: May 2005

A workshop entitled Cellular Protein Databases from Two‐Dimensional Gel Electrophoresis was held in Atlanta, Georgia, 28 February–1 March 1987. Its purpose was to assess the status of two‐dimensional gel electrophoresis of proteins as a research methodology in biological systems, particularly in the generation of cellular protein databases. The workshop participants summarized current studies on a variety of biological systems, both prokaryotic and eukaryotic. Analysis of the progress being made led to the conclusion that electrophoretic techniques, supported by automatic scanning of gel images and computer‐assisted processing, analysis and matching of gel images, are now capable of generating databases of great potential value. Factors affecting the reproducibility of protein spot patterns on gels were identified, and the extent to which gel pattern variability causes difficulties in communicating results and in integrating information from different laboratories was assessed. Measures were suggested to help overcome obstacles to the generation of comprehensive cellular protein databases from the electrophoretic resolution of total cellular proteins.
Computerized, comprehensive databases of cellular and secreted proteins from normal human embryonic lung MRC‐5 fibroblasts: Identification of transformation and/or proliferation sensitive proteins
Authors: Celis Julio E. , Ratz Gitte P. , Madsen Peder , Gesser Borbala , Lauridsen Jette B. , Hansen Karl P. Brogaard , Kwee Sianette , Rasmussen Hanne Holm , Nielsen Henrik V. , Crüger Dorthe , Basse Bodil , Leffers Henrik , Honore Bent , Møller Olaf , Celis Ariana
Published: May 2005

Databases of protein information from human embryonal lung fibroblasts (MRC‐5) have been established using computer analyzed two‐dimensional gel electrophoresis. One thousand four hundred and eighty‐two cellular proteins (1060 with isoelectric focusing and 422 with nonequilibrium pH gradient electrophoresis, in the first dimension) ranging in molecular mass between 8 and 234 kDa were separated and numbered. Information entered in the database (in most cases for major proteins) includes: protein name, HeLa protein catalog number, mouse protein catalog number, proteins matched in transformed human epithelial amnion cells (AMA) and peripheral blood mononuclear cells (PBMC), transformation and/or proliferation sensitive proteins, synthesis in quiescent cells, cell cycle regulated proteins, mitochondrial and heat shock proteins, cytoskeletal proteins and proteins whose synthesis is affected by interferons. Additional information entered for a few transformation‐sensitive proteins that have been selected for future studies includes levels of synthesis and amounts in fetal human tissues. A total of four hundred and seventy‐six [35S] methionine labeled polypeptides (258 isoelectric focusing; 218, nonequilibrium pH gradient electrophoresis) secreted by MRC‐5 fibroblasts were separated and recorded (J. E. Celis et al., Leukemia 1987, 1, 707–717). Information entered in this database includes molecular weight and transformation sensitive proteins. These databases, as well as those of epithelial and lymphoid cell proteins (J. E. Celis et al., Leukemia 1988, 9, 561–601), represent the initial stages of a systematic effort to establish comprehensive databases of human protein information. In the long run, these databases are expected to offer a useful framework in which to focus the human genome sequencing effort.