bluuebunny/crossref_metadata_2025_split huggingface dataset:
Isoelectric focusing analysis of antibody clonotype changes occurring during immune responses using immobilized pH gradients
Authors: Knisley Keith A. , Rodkey L. Scott 
Published: May 2005

AbstractSerum was collected from rabbits at 2‐day intervals following a single injection with tetanus toxoid or at weekly intervals following multiple injections with Micrococcus lysodeikticus cell walls. These sera were analyzed for the presence of individual clonotypes of specific anti‐tetanus or anti‐micrococcal antibodies by isoelectric focusing in immobilized pH gradients with added carrier ampholytes followed by affinity immunoblotting. The affinity immunoblots obtained clearly defined both the rapid disappearance and late appearance of distinct subsets of antibody clonotypes during the response. These data demonstrate the application of affinity immunoblotting combined with immobilized pH gradients for detecting the subtle changes in specific antibody clonotype patterns which occur during an immune response.
Isoenzyme analysis of lichen algae in immobilized pH gradients
Authors: Kilias Harald , Gelfi Cecilia , Righetti Pier Giorgio 
Published: May 2005

AbstractA base for a modern species' concept of chlorococcal algae can be obtained, not by morphological analysis, but by biochemical characters, e. g. isoenzyme banding patterns. From isolated lichen algae of the genus Trebouxia de Puymaly a set of five such enzymes has been studied by isoelectric focusing in immobilized pH gradients (IPG): phosphoglucomutase, phosphoglucose isomerase, malate dehydrogenase, mannitol dehydrogenase and leucine aminopeptidase. The first four are resolved into isoforms in a pH 4–7 IPG interval, while the last one is analyzed in an IPG pH 3.5–5 span. The patterns are specific for distinct populations, inter‐ and intraspecifically varying in dependence from their geographical distribution or the lichen species from which they have been isolated. Their limited heterogeneity (one to four isoforms) suggests that they are the products of specific genes rather than artefacts of the extraction procedure or the IPG analysis. Sharp isozyme patterns can only be obtained in a mixed‐bed, carrier‐ampholyte (CA)‐IPG gel and by anodic application, suggesting that the recently proposed mechanism of hydrophobic protein‐IPG matrix interaction (Electrophoresis, 1987, 8, 62–70) is fully operative here. As an additional mechanism, it is proposed that, in some cases, CA might simply act, when added to an IPG gel, by buffering, in the transient state, the sample zone before the protein migrates from the liquid phase into the IPG matrix.
Radically simplifying the Henry function
Authors: Michov Budin M. 
Published: May 2005

AbstractWe recently established that every ion has two radii – a geometrical one and an electrokinetic one. Using this principle, and in accordance with Henry's method of determining his function, f(κa), we have discovered that the Henry function can be radically simplified. The graphs of the proposed equation are plotted at different argument values. They are identical with the graphs plotted according to the Henry function.
Purification of cellulases from Streptomyces strain A20 by electroendosmotic preparative electrophoresis
Authors: Curioni Andrea , Peruffo Angelo Dal Belin , Nuti Marco P. 
Published: May 2005

AbstractFive cellulase components were identified and purified in one step from Streptomyces strain A20 using electroendosmotic preparative electrophoresis. By this procedure up to 18 mg of protein mixture could be loaded on the column, with an estimated recovery of 60–70% of total activity; activity and protein recovery could be estimated 32% and 47%, respectively, if only activity peaks were considered. In comparison to other purification methods, this technique results in high protein recovery and resolution of applied samples.
Rapid imaging, using a cooled charge‐coupled‐device, of fluorescent two‐dimensional polyacrylamide gels produced by labelling proteins in the first‐dimensional isoelectric focusing gel with the fluorophore 2‐methoxy‐2,4‐diphenyl‐3(2H)furanone
Authors: Jackson Peter , Urwin Valerie E. , MacKay Craig D. 
Published: May 2005

AbstractA new method for visualising proteins in two‐dimensional polyacrylamide gels was developed. Proteins were labelled with the fluorophore 2‐methoxy‐2,4‐diphenyl‐3(2H)furanone (MDPF) while present in the first‐dimensional gel after isoelectric focusing and subsequently electrophoresed into the second‐dimensional gel. High resolution spot patterns were produced and compared with other methods of visualisation. A new rapid imaging system based on a cooled charge‐coupled‐device was used to view the two‐dimensional fluorescent protein spot patterns. The method allows the immediate and rapid imaging of two‐dimensional gels at the end of electrophoresis with no further processing.
Characterization of human skin fibroblast extracellular proteins by two‐dimensional polyacrylamide gel electrophoresis
Authors: Graham Colin A. , McLean W. H. Irwin , Hughes Anne E. , Nevin Norman C. 
Published: May 2005

AbstractHuman skin fibroblasts secrete over 50 proteins into the culture medium. In this paper these are characterised using two‐dimensional polyacrylamide gel electrophoresis and peptide mapping of proteins metabolically labelled in the presence and absence of tunicamycin. Thirty of these proteins have been shown to be N‐glycosides, 4 are O‐glycosides and 10 are not glycosylated. Of the major proteins, groups 1–4 have previously been shown to be fibroblast specific. Peptide mapping and tunicamycin treatment has identified that groups 1 and 2, and 3 and 4 are closely related and that groups 1 and 3 arise by N‐glycosylation of 2 and 4, respectively. The unglycosylated precursor forms of several other proteins have also been identified. This approach to the analysis of protein secretion provides an abundance of information on many proteins simultaneously and can be used to assess the changes in protein secretion associated with development, and to identify extracellular growth factors and other regulatory proteins.
Characterization of genetic variants of human serum transferrin by isoelectric focusing: Comparison between conventional and immobilized pH gradients, and application to a protocol for paternity testing
Authors: Pascali Vincenzo L. , Dobosz Marina , Destro‐Bisol Giovanni , D'Aloja Ernesto 
Published: May 2005

AbstractThe polymorphism of transferrin (Tf) is currently being studied by isoelectric focusing in carrier ampholyte‐generated pH gradients, carrier ampholyte‐separator pH gradients or in immobilized pH gradients. Details for obtaining reproducible results with each of the three procedures are outlined. The effectiveness of pretreatment of serum samples with ferrous/ferric salts is discussed, and incubation times optimized after spectrophotometric measurement of the monoferric Tf conversion. Most of the presently available commercial batches of carrier ampholytes do not reliably discriminate the six common TfC subtypes. Resolution of C1, C3 and C2 was achieved by adding 20 to 90 mM HEPES slab gels prepared with various carrier ampholytes. Isoelectric focusing in carrier ampholyte‐separator pH gradients cannot be recommended as a standard typing procedure because the results strongly depend on the batch of carrier ampholytes. Tf subtype resolution was only achieved by using isoelectric focusing in immobilized pH gradients with pH slopes reliably reproducible from one experiment to another. Two major shortcomings of immobilized pH gradients are a marked tendency to protein precipitation at the application site and an interaction of proteins with the charged matrix. A protocol for Tf subtyping in immobilized pH gradients is described, based on prior desialylation of samples instead of pretreatment with iron. Sample entry into the matrix was optimized by addition of 5 mM Tris to the gels, and initially running them at low voltage. Recommendations are provided for the application of Tf typing for paternity testing.
Factor B(BF) subtyping by isoelectric focusing: Methods, nomenclatures, genetics and forensic application
Authors: Geserick Gunther , Patzelt Dieter 
Published: May 2005

AbstractUsually factor B (BF) typing is performed by means of the traditional agarose gel electrophoresis. Using isoelectric focusing, the system can be extended by two common subtypes of BF F. The existence of BF F subtypes has in the meantime been confirmed by various authors and in different populations. Their inheritance has been proven by family‐and mother/child analyses and molecular‐genetic studies (correlation with restriction fragment length polymorphism). Different typing methods as well as different nomenclatures seem to indicate that the subtypes FA and FB (according to Gesericket al.) are identical with the Fb and Fa subtypes (according to Teng and Tan). At present, some confusion still exists for the less frequent variants and subtypes which possibly could be identified by direct comparison of the patterns. The BF system is a valuable marker in patennity testing. Its chance for exclusion of paternity in Caucasian populations has been calculated to be about 14 % for agarose gel electrophoresis and increases to about 16 % for BF F subtyping by isoelectric focusing. Preliminary results indicate that BF may also be used for typing of bloodstains (up to 2 weeks old).
A new sample applicator for isoelectric focusing in horizontal polyacrylamide gels
Authors: Altland Klaus , Becher Pia 
Published: May 2005

AbstractA new sample applicator for horizontal flat gels is described. The applicator is practically safe against contamination from adjacent samples and can be used for all types of electrophoretic separations including a concentration step for either the sample (i. e.disc electrophoresis) or the separated zones (i. e.isoelectric focusing). The applicator is a piece of flat glass with 26 or 51 parallel 2 mm wide grooves, drilled at distances of 9 or 4.5 mm. Samples, maximally 25 or 50. are applied to the areas between the grooves. By inverting the applicator, the samples are brought into close vicinity to the gel surface and the pendant droplets expand by capillary attraction into the slits between the glass and gel with resultant even distribution across the lanes of 2.5 or 7 mm width. The applicator can be used for separations with and without protection of the electrophoretic setup by paraffin oil and allows for fast multiple handling of samples by means of appropriate syringes and microtiter plates.
Electroblotting of Immobiline DryPlates applied to identification of human plasma apolipoprotein A‐I
Authors: Holmquist Leif 
Published: May 2005

AbstractA method for efficient electroblotting of Immobiline DryPlates, allowing subsequent immunological identification of separated proteins has been developed. A thin layer of 1 % agarose containing sodium dodecyl sulfate is moulded on the 0.5 mm thick polyacrylamide gel surface after completed electrophoresis. After separation of the agarose‐polyacrylamide gel sandwich from the plastic film the rigid gel sandwich could be easily transferred to a nitrocellulose membrane and electroblotting could be performed without adherence of the sticky polyacrylamide gel layer to the membrane. Using this technique human plasma high density apolipoprotein A‐Iisoforms, over a wide concentration range, could be identified in a heterogeneous mixture, conserving the isoform pattern and band sharpness produced in the immobilized pH gradient experiments.
Immobilized pH gradients in ultrathin‐layer (100 μm) gels: Avoiding capillarity by a ‘squeezing‐sealing mold’ technique
Authors: Pascali Vincenzo L. , Dobosz Marina , D'Aloja Ernesto 
Published: May 2005

AbstractSeveral procedures for casting 100 μm ultrathin immobilized pH gradients are described. When acrylate/glass molding cassettes (Pascali et al., Electrophoresis 1987, 8, 371–373) are used two main problems are encountered: (i) a tendency of polymerization solutions, at the beginning of delivery, to spread across the glass surfaces with troublesome effects on the gradient stratification, and (ii) the raising of steep menisci at both extremities of the pH intervals, originating from capillarity phenomena and resulting in nonuniform gradients with bowed electrophoretic patterns. The first shortcoming was acceptably solved by increasing the density of sucrose gradients, and pouring them into prewarmed molding cassettes. The detrimental effect of menisci could be overcome by using a ‘squeezing‐sealing mold’ technique. A molding cassette was endowed with a continuous, squared spacer frame, the upper side being open by inserting a wedged clip. A slight excess of polymerization solution was first dispensed into the cassette and squeezed away on removal of the clip. By completely excluding air from the molding cassette, uniform and well reproducible ultrathin gels could be cast. A major advantage of ultrathin immobilized pH gradient gels is the drastically shorter focusing time.
Two‐dimensional electrophoresis. Two‐dimensional gel electrophoresis of platelet polypeptides with immobilized pH gradients in capillary tubes
Authors: Asakawa Jun‐ichi 
Published: May 2005

AbstractTwo‐dimensional electrophoresis (2‐DE) with immobilized pH gradient (IPG) gels in capillary tubes was used in the first‐dimensional isoelectric focusing (IEF) for the separation of human platelet polypeptides. Two types of IPG tube gels, pH ranges 4–8 and 7–10, containing 8 M urea, 1% Nonidet P‐40 and 0.1% pH 3.5–10 Ampholine carrier ampholytes (CA) were prepared by a simple method not requiring special equipment. The addition of CA to both gel and sample solutions was essential in the tube gel IPG system. Proteins were visualized by a modification of Wray's silver‐staining technique. The degree of resolution and the number of spots observed on an IPG 2‐DE gel with pH 4–8 were comparable with those obtained with O'Farrell's high‐resolution 2‐DE. Approximately 200 basic polypeptides, which are difficult to separate by conventional CA‐based IEF 2‐DE or the non‐equilibrium pH gradient system, were well resolved by 2‐DE with a pH 7–10 IPG tube gel in the first‐dimension. The gel patterns with either pH gradient 4–8 or 7–10 were highly reproducible among gels prepared and run simultaneously. These results demonstrated the potential and usefulness of the 2‐DE system with IPG gels in capillary tubes.
Microheterogeneity of human plasma lecithin:cholesterol acyltransferase examined by isoelectric focusing in immobilized pH gradients
Authors: Holmquist Leif , Bjellqvist Bengt 
Published: May 2005

AbstractThe microheterogeneity of highly purified human plasma lecithin:cholesterol acyltransferase (LCAT) has been examined by electrophoresis in immobilized pH gradients in Immobiline‐polyacrylamide gels of the pH ranges 4–7 and 4.2–4.9. Seven isoforms were obtained with LCAT isolated from pools of normal plasma. Using this technique the apparent pI values at 15 °C for the isoforms in the pH 4.2–4.9 gradient were 4.37, 4.42, 4.48, 4.53, 4.60, 4.67 and 4.74. (SD = ± 0.03 for all). The most intensely stained band in the isoform pattern corresponded to the isoform with a pI value of 4.48.
Immobilized pH gradient isoelectric focusing of cerebrospinal fluid proteins
Authors: Pirttilä Tuula , Frey Harry , Mattila Kari , Sidén Åke 
Published: May 2005

AbstractIsoelectric focusing in immobilized pH gradients, supplemented with 0.5 % w/v carrier ampholytes was applied for studies of native proteins, especially immunoglobulin G, in cerebrospinal fluid and serum. All 72 paired samples were run on pH 4–10 gels; 25 of them were also examined in pH 7–10 gels. Silver staining and nitrocellulose blotting with amplified immunoperoxidase detection of immunoglobulin G were used for protein visualization. Intrathecally produced immunoglobulin G was resolved into sharply focused, straight and easily identifiable fractions. The pH gradients were stable and the inter‐gel reproducibilities of individual immunoglobulin G patterns were good.
Subtyping of group specific component (GC) in human semen, blood and vaginal fluid by isoelectric focusing in immobilized ph gradients
Authors: Pötsch‐Schneider Lucia , Klein Hartmut 
Published: May 2005

AbstractThe group specific component (GC) is stable and well suited for forensic casework. Isoelectric focusing of common GC variants from semen, seminal fluid, vaginal fluid and semen stains, on Immobiline DryPlates, pH 4.5–5.4, is of practical value in criminal investigations of sexual deliquencies. GC is present in normospermia and azoospermia seminal fluids and found in about 20 % of the vaginal secretions. The GC patterns observed were similar and in accordance with the bands of the individual GC type in plasma/serum.
Application of immobilized pH gradient isoelectric focusing to forensic hemogenetics: A survey on a three year experience with the transferrin (TF) and alpha 1‐antitrypsin (PI) systems
Authors: Skoda Ulrike , Faßbender Lydia , Händler Christa , Mauff Gottfried , Pulverer Gerhard 
Published: May 2005

AbstractThe subtypes of transferrin (TF) and alpha 1‐antitrypsin (PI), first discovered using isoelectric focusing, are now mostly determined in immobilized pH gradient gels. We report on our experience in the parentage expertise with both polymorphisms over a period of three years. The complexity of the technology was compensated by the fact that most subtypes of TF and PI could be more reliably recognized. The PI alleles PI*M1, M2, M3, S, F, T, and Z and TF alleles TF*C1, C2 and C3, and in addition four further rare TF alleles were observed. The allele frequencies from non‐related individuals did not deviate from the Hardy‐Weinberg equilibria and corresponded well to known frequencies from West Germany and other Caucasoid populations. With the TF system 36 accused men, and with the PI system 54 were excluded from paternity from a total of 344 (TF) respectively 347 (PI) cases. From the data presented here isoelectric focusing in immobilized pH gradient gels appears to be a major improvement over carrier ampholyte generated pH gradients in the distinction of TF and PI phenotypes.
Isoenzyme patterns as a tool in taxonomy of chlorococcal algae
Authors: Kilias Harald 
Published: May 2005

AbstractFrom isolated lichen algae of the genusTrebouxia, De Puymaly isoenzymes of phosphoglucomutase, malate dehydrogenase and superoxide dismutase were studied by isoelectric focusing in hybrid immobilized pH gradients. Phosphoglucomutase has proven to be a suitable marker since it is highly variable whereas malate dehydrogenase and superoxide dismutase are less variable. The focusing patterns are species specific and provide a basis for a reevaluation of species classification ofTrebouxia.
Genetic polymorphisim of a lymphocyte protein (p75) with six different alleles studied by two‐dimensional electrophoresis: Qualitative and quantitative data
Authors: Waldinger Dorothea , Cleve Hartwig 
Published: May 2005

AbstractThe genetic polymorphism of a human lymphocyte protein (p75) was studied by two‐dimensional electrophoresis within 17 families and, in addition, 22 unrelated individuals from Southern Germany, resulting in a total of approximately 100 individuals, The cytosolic and membrane proteins from cell lysates of phytohemagglutinin stimulated and [3H] leucine labeled lymphocytes were separated by two‐dimensional electrophoresis. The p75 protein with an approximate molecular weight of 75 000 occurred in six variants with slightly different isoelectric points and/or apparent molecular weights. Three common variants (a, b, and c) and three rare variants (d, e and f) could be distinguished. Among the approximately 100 individuals studied we observed 15 different phenotypes, three homozygous (p75‐a, ‐b, ‐c) and 12 heterozygous (p75‐ab, ‐ac, ‐bc, ‐ad, ‐ae, ‐be, ‐bf, ‐cd, ‐ce, ‐cf, ‐de, ‐df) phenotypes. The genetics of the p75 protein variations was ascertained by family studies and quantitative computer analysis. We were able to show a Mendelian mode of inheritance of the variants within the families and a gene‐dosage dependence of the protein spots in homozygous and heterozygous phenotypes. The data allowed us to assume a polymorphic protein p75 determined by six alleles on a autosomal gene locus. The allele frequencies were calculated from the phenotype distribution within 56 unrelated individuals. The gene frequencies of the three common alleles ranged between 0.38 and 0.22 and the gene frequencies of the three rare alleles ranged between 0.01 and 0.07.
The use of ultrathin‐layer polyacrylamide gel isoelectric focusing in two‐dimensional analysis of plant and fungal proteins
Authors: Marlow Gary C. , Wurst Denise E. , Loschke David C. 
Published: May 2005

AbstractAlthough use of ultrathin polyacrylamide gel isoelectric focusing in the first dimension of two‐dimensional analysis bestows a number of advantages, it has been little used by the plant science community. Nonstandardization along with problems unique to the format have probably delayed wider adoption. Relevant parameters were therefore tested in order to optimize resolution, reproducibility, economy and ease of use. Ultrathin‐layer gels (200 μm in this study) used in the first dimension require a semirigid backing for support. Widely available matte‐finished thin polyester film without chemical pretreatment was found to bind the gel adequately. The gel adheres to the film through all processing steps, yet, if desired, can be easily transferred to Whatman 3MM paper for special applications such as Western blotting. The ultrathin first‐dimensional gels can be quickly dried on the polyester backing for convenient handling and long term storage. Strips cut from the dried gel for use in the second dimension are more easily manipulated than their tube format counterparts. The difficulty of disrupting and recovering microsamples of labeled leaf and root tissue prompted the invention of an efficient and simple communition device. An economical and efficient silver stain process is also described. This analytical technique was applied in an attempt to detect resistance gene products in different genetic backgrounds of maize. Although the ultrathin flatbed format provides as good as or better resolution than the tube gel system, the level of sensitivity was still inadequate to reveal the apparently rare resistance gene product.
Significant immunological cross‐reactivity of plant glycoproteins
Authors: Laine Anne‐Catherine , Faye Loïc 
Published: May 2005

AbstractPlant glycoproteins generally cross‐react because of the presence of identical or related complex glycans which are highly immunogenic. The use of mild periodate oxidation of glycans after glycoprotein transfer from sodium dodecyl sulfate‐poly‐acrylamide gel electrophoresis gels to nitrocellulose membranes prior to immunodetection is a way of identifying the carbohydrate antigenic determinants of a glycoprotein as the basis for antigenic cross‐reaction. Periodate oxidation can distinguish between antibodies directed against carbohydrate and against peptide antigenic determinants, the latter being unaffected by oxidation. Immunoblotting performed after periodate treatment allows the detection of common protein epitopes.
Multi‐chamber apparatus for preparative isoelectric focusing
Authors: Kyhse‐Andersen Jan 
Published: May 2005

AbstractA multi‐chamber apparatus for preparative isoelectric focusing is described. The apparatus is constructed of 32 separation chambers and 2 electrode chambers, all separated by uncharged porous membranes. The total volume of the 32 separation chambers is 660 mL. A cooling system and a stirring system are built in. Human serum proteins were separated by isoelectric focusing in a natural pH gradient. The fractionation was monitored by fused rocket immunoelectrophoresis. The number of fractionation was monitored by fused rocket immunoelectrophoresis. The number of proteins in each fraction was monitored by crossed immunoelectrophoresis. The apparent pI values of IgG. transferrin and alpha‐1‐antitrypsin are as found in the literature. Orosomucoid (alpha‐1‐acid glycoprotein) (pI = 1.8) is concentrated at the acid end of the pH gradient.