bluuebunny/crossref_metadata_2025_split huggingface dataset:
Fast isoelectric focusing of milk proteins on small ultrathin polyacrylamide gels containing urea
Authors: Moio Luigi , Luccia Aldo Di , Addeo Francesco 
Published: May 2005

AbstractThe preparation of 0.45 mm thin polyacrylamide gels, containing urea, for horizontal micro isoelectric focusing of milk proteins with PhastSystem is described. Isoelectric focusing in the small gels, stained either with Coomassie Brilliant Blue R‐250 or with the more sensitive silver stain, affords a fast and sensitive procedure for an analysis of milk and cheese proteins. The procedure can be effectively exploited in detecting adulteration in ovine cheese with bovine milk.
Diagnostic use of an analysis of urinary proteins by a practicable sodium dodecyl sulfate‐electrophoresis method and rapid two‐dimensional electrophoresis
Authors: Lapin Alexander , Gabl Franz , Kopsa Herbert 
Published: May 2005

AbstractTwo methods suitable for routine clinical analyses of urinary proteins are presented and compared. The first is a horizontal sodium dodecyl sulfate‐polyacrylamide gel electrophoresis technique, suitable for simultaneous analysis of 20 native urinary samples. This method uses polyacrylamide gradient gels, prepared with a laboratorybuilt gel casting device. The second method is a rapid two‐dimensional electrophoresis procedure, combining cellulose acetate electrophoresis and sodium dodecyl sulfate‐electrophoresis. The first step uses a routine system (Chemetron), the second separation step followed by staining with Coomassie Brilliant Blue R is performed on the PhastSystem. The resulting two‐dimensional patterns reveal urinary proteins distributed according to the 5‐zone pattern of native proteins (albumin, alpha‐1, alpha‐2, beta, gamma‐globulin) as well as to the logarithm of their molecular weights. Examples of (routine) diagnoses with a special interest in the monitoring of kidney transplant patients are shown.
Reactivities of immunoglobulin E and immunoglobulin G subclasses identified by isoelectric focusing‐immunoprint in allergic patients
Authors: Becker Wolf‐Meinhard 
Published: May 2005

AbstractProbing of IgE or IgG subclass reactivities on single allergenic components in an extract is tedious and time‐consuming under native conditions. Isoelectric focusing immunoblotting combines a powerful, highly resolving native separatior method with the specificity and sensitivity of immunological test methods. This method is easy and quick to perform. The potential usefulness of this method is demonstrated by eight examples of a type I or type III allergy analyzing IgE and IgG subclass reactivities and their distribution.
Enzyme‐linked immunoglobulins and their clinical significance
Authors: Tozawa Tatsuo 
Published: May 2005

AbstractMost of the enzymes used in routine biochemical tests have been detected in circulating enzyme‐linked immunoglobulin (Ig) complexes (E‐Ig). The complexes often cause unexplained hyperenzymemia and give anomalous patterns on isozyme electrophoresis. However, E‐Ig does not appear to be associated with any pathological conditions. The present study was undertaken to determine the clinical characteristics of patients with E‐Ig. More than 42 000 patients selected at random and 10 000 blood donors were screened for lactate dehydrogenase (LDH)‐linked Ig complexes (LDH‐Ig), amylase (Amy)‐linked Ig complexes (Amy‐Ig), alkaline phosphatase (AP)‐linked Ig complexes (AP‐Ig) and creatine kinase (CK)‐linked Ig complexes (CK‐Ig). LDH‐Ig and Amy‐Ig were screened by electrophoresis for routine isozyme analysis and identified by precipitin reactions. AP‐Ig and CK‐Ig were screened and identified by counter immunoelectrophoresis. The incidence of all E‐Ig ranged from 0.18 % to 0.61 % in patients and from 0.03 % to 0.23 % in blood donors. The incidence curves of E‐Ig were age‐related, generally increasing with age. The curves of E‐IgG's for different enzymes resembled each other in shape but those of E‐IgA's did not. A positive correlation between E‐IgG and autoimmune diseases was present. Consequently, it is concluded that both autoimmune diseases and age are the common clinical factors involved in E‐IgG. No specific diseases were found for cases with E‐IgA. However, it is suggested that some CK‐IgA, but not all, is caused by elevated serum CK activity.
Rehydratable agarose gels: Application to isoelectric focusing in 9 molar urea
Authors: Hoffman Wayne L. , Jump Adrien A. , Kelly Patrick J. , Elanogovan Natarajan 
Published: May 2005

AbstractA method is described for the preparation of rehydratable agarose gels, with specific application to the direct incorporation of 9 M urea and carrier ampholytes into rehydratable agarose gels for use in isoelectric focusing. After drying the agarose gel containing an uncharged linear polyacrylamide, one gel volume of a 9 M urea‐carrier ampholyte solution is absorbed directly into the gel in 60 min, eliminating equilibration or dialysis of the gel in larger volumes of the 9 M urea‐carrier ampholyte solution. Proteins with a molecular mass of 970 000 Da can be separated by isoelectric focusing in these rehydratable gels. The focused proteins can then be quantitatively transferred to nitrocellulose in less than 10 min, and any immunostaining procedure can be used to probe the blotted proteins. These agarose gels are easy to make, they rehydrate rapidly and they can be used in applications other than isoelectric focusing.
Isoelectric focusing of membrane proteins: High resolution separation of myelin proteins
Authors: Persson Håkan , Corneliuson Olle 
Published: May 2005

AbstractA mixture of the nonionic detergent Triton X‐100, the zwitterionic detergent 3‐[(cholamidopropyl)dimethylammonio]‐1‐propanesulphonate (CHAPS), 9M urea and carrier ampholytes was found comparable to media containing sodium dodecyl sulfate in the capacity for solubilization of myelin proteins, including the highly hydrophobic proteolipid protein. The solubilized sample was incorporated into the polymerization mixture before moulding an ultrathin gel, with heat convection characteristics allowing a high wattage to be applied, thus allowing fast separation with high resolving power. Since the most basic protein in myelin focuses at a pH > 10, fast separation is essential in order to minimize decay of the cathodic end of the pH gradient.
Enantiomer resolution in immobilized pH gradient gels via inclusion of a chiral discriminator
Authors: Righetti Pier Giorgio , Ettori Claudia , Chafey Philippe , Wahrmann Juan Pedro 
Published: May 2005

AbstractThe separation of enantiomeric forms of dansylated amino acids by isoelectric focusing in immobilized pH gradients (IPG) is demonstrated for the first time. Separations occur in a pH 3.0–4.0 IPG interval, in presence of 7murea, 10% methanol and 60 mM β‐cyclodextrin (CD) as chiral discriminator. It is found that the inclusion complex formed between the D‐form and CD has a lower pI than the uncomplexed form (ΔpI = 0.05 for DL‐Phe and ΔpI = 0.025 for DL‐Trp); from this, it is calculated that the pK of the tertiary amino group in the dansyl moiety is lowered by 0.1 pH unit in the former case (D‐Phe) and by 0.05 in the case of D‐Trp (both values referring to 60 mM CD gels). For some racemates (e.g., DL‐Phe) the separation mechanism is still operative with CD concentrations as low as 20 mM. In our system 60 mM CD appears to be the solubility limit of CD. As the complex is stable in the electric field for at least 15 h, this separation mechanism could be exploited for purifying large quantities of pure D and L forms racemates in multicompartment electrolyzers with isoelectric Immobiline membranes.
Two‐dimensional polyacrylamide gel electrophoresis of extracellular soybean pathogenesis‐related proteins using PhastSystem
Authors: Roggero Piero , Pennazio Sergio 
Published: May 2005

AbstractAcidic and basic pathogenesis‐related proteins (PR‐Ps) were extracted from the intercellular fluid (IF) of soybean leaves, locally infected with tobacco necrosis virus and showing necrotic local lesions. Proteins were detected by two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE)using PhastSystem and precast commercially available gels. Extracts from healthy leaves were run as controls. PR‐Ps were first run under native PAGE conditions or isoelectric focusing (IEF), the gels stained with Coomassie Blue, then run under sodium dodecyl sulfate (SDS)‐denaturing conditions and finally stained with silver. Ten major acidic PR‐Ps were separated; their Mr's were close to those found by conventional PAGE. Their isoelectric points ranged from 3.5 to 5.0. Ten basic PR‐Ps were separated and their Mr's estimated. None of these acidic or basic soybean PR‐Ps was a glycoprotein. PAGE with Phast‐System and precast gels gives reliable results, comparable with those from conventional 2D‐PAGE, with simpler experimental procedures. By electrophoresing Coomassie‐stained gels with SDS in the second dimension, we were able to control the first‐dimensional separation and to avoid laborious protocols generally adopted with unstained gels.
Suitability of staining techniques for the detection and quantitation of nonhistone high mobility group proteins
Authors: Csordas Adam , Pedrini Michael , Grunicke Hans 
Published: May 2005

AbstractThree different staining techniques were compared for the detection of nonhistone high mobility group (HMG) proteins after acidic urea‐polyacrylamide gel electrophoresis [1]. Silver staining after glutaraldehyde fixation [2] provides the highest detection sensitivity. Because of the acid solubility of HMG proteins special care has to be taken concerning fixation. Staining with colloidal CBB G‐250 according to Neuhoff et al. [3] is superior in sensitivity and reliability of quantitation when compared with noncolloidal Coomassie Brilliant Blue R‐250. High detection sensitivity and reproducibility of quantitation are prerequisites for studying the tissue‐specific expression of HMG proteins. In the present study tissue‐specific differences in the molar amounts of various HMG proteins in thymus and erythrocytes of the chicken are documented by application of the methods tested.
Electrofocusing of acid phosphatases from frog liver, using an immobilized pH gradient
Authors: Kubicz Aleksandra , Szalewicz Agata , Fawcett John S. , Chrambach Andreas 
Published: May 2005

AbstractIsoelectric focusing on carrier ampholyte‐containing immobilized pH gradient gels was applied (i) to gels submerged in silicone oil on a Peltier cooled apparatus, (ii) to the separation of the higher molecular weight (HMW, Mr 140 000) and the lower molecular weight (LMW, Mr 38 000) acid phosphatases (AcPases) from frog livers. (i) Electrofocusing was conducted on gels submerged under silicone oil cooled and stirred on a Peltier‐thermoregulated horizontal gel support plate. This procedure aimed at (a) improving the temperature control of the gel by direct contact of coolant with the gel surface, and thus at being able to focus at the maximal field strength and consequently highest resolution; (b) preventing evaporation from the gel and (c) excluding atmospheric carbon dioxide. Silicone oil submersion did not abolish water loss from the gel into the electrolyte strips during isoelectric focusing, or a rippled gel surface. Absence of water exudation on the ripples noted previously by Altland [1] was observed. (ii) The electrofocusing of AcPases on immobilized pH gradients yielded patterns which remained stationary as a function of time, by contrast to previous analyses on carrier ampholyte generated pH gradients. The total number of enzymatically active components found in the enzyme preparations from different stages of purification and in the isolated HMW and LMW AcPases was 18. The HMW and LMW AcPases focused in characteristic pH ranges and exhibited qualitative and quantitative pattern differences. Their band patterns add up to that of a crude preparation containing both enzymes. Neither polyacrylamide gel electrophoresis (PAGE) at any nondenaturing pH, nor isoelectric focusing in carrier ampholytes with pattern changes due to the pH gradient drift were able to yield that result.
Major histocompatibility complex (MHC) protein analysis by optimised two‐dimensional electrophoretic methods
Authors: Fernandez Nelson , Labeta Mario , Kurpisz Marciej , Nakatsuji Takako , Sachs John , Festenstein Hilliard 
Published: May 2005

AbstractHuman histocompatibility molecules HLA‐Class I and Class II (DR, DQ, DP) were analysed using three two‐dimensional protocols: nonequilibrium pH gradient electrophoresis (NEPHGE), isoelectric focusing‐acidic gradient (IEF‐AG) and isoelectric focusing‐basic gradient (IEF‐BG). The three methods differ in their carrier ampholyte combinations and electrophoretic conditions. They provide different pH gradients and therefore different electrocusing profiles. The NEPHGE protocol was edequate for separating proteins across a broad range of pI mobilities, i. e. 4.4. pH units between the acidic and the basic end. In contrast, the IEF‐AG and the IEF‐BG protocols gave a sepration power across a narrow pH range, 1.9 and 1.7 pH units respectively. Thus, whereas the NEPHGE protocol provides a tool for a global major histocompatibility complex (MHC) antigen profile analysis, the IEF‐AG and ‐BG allows one to investigate subcomponents of the individual MHC chains. For example, NEPHGE analysis of the HLA Class I heavy chain revealed a single spot. However, IEF‐BG revealed the presence of six equidistantly spaced spots spanning a short pH gradient with identical molecular weight. Similar improved resolution was seen for the HLA‐DR, DQ, and DP molecules. The IEF acidic gradient was adequate for separating the alpha chain; the IEF basic gradient gave better resolution of the beta chains. This data provides a baseline set of conditions for both analytical and preparative MHC protein studies prior to amino acid sequencing.
Towards establishing a protein database of Drosophila
Authors: Santarén Juan F. 
Published: May 2005

AbstractAn improved method of high‐resolution two‐dimensional gel electrophoresis has been used to study the patterns of protein synthesis in wing imaginal discs of late instar larvae of Drosophila melanogaster. A total of one thousand and twenty five labelled polypeptides (787 acidic and 238 basic) have so far been separated and catalogued. For convenience, all these polypeptides have been numbered and their position fixed by its molecular weight and relative mobility. They are indicated on a reference protein map for further studies.
Genetic studies of human apolipoproteins: XIV. A simple agarose isoelectric focusing gel method for apolipoprotein E phenotyping
Authors: Kamboh Mohammad I. , Kelly Lori J. , Ferrell Robert E. 
Published: May 2005

AbstractA new and simplified procedure is described for apolipoprotein E (APO E) phenotyping from native plasma or serum samples. Diluted or dialyzed samples are separated on agarose isoelectric focusing gels followed by protein blotting on nitrocellulose membranes. APO E banding patterns are localized immunologically using polyclonal goat anti‐APO E antiserum as the primary antibody and rabbit anti‐goat IgG conjugated with alkaline phosphatase as the secondary antibody. The method was used in parallel with our previously described polyacrylamide gel system to screen 110 unrelated and healthy US whites. Both gel systems gave identical APO E phenotypes, and allele frequencies were comparable with reported US white values. This simplified method can be used on a large number of population and clinical samples with minimum cost and effort.
Two‐dimensional electrophoresis of heart muscle proteins in human cardiomyopathies
Authors: Kovalyov Leonid I. , Naumov Vladimir G. , Pulyayeva Helena V. , Samko Anatoli M. , Tsvetkova Marina N. , Shishkin Sergei S. , Mukharlyamov Nurmukhmmed M. 
Published: May 2005

AbstractHuman heart muscle proteins have been analyzed by two‐dimensional electrophoresis. Twenty five autopsy heart muscle samples obtained from individuals who had died in accidents and who had no signs of cardiovascular pathology have been compared with biopsy and autopsy myocardium samples of patients with: dilated cardiomyopathy (5 cases), hypertrophical cardiomyopathy (2 cases) and myocarditis (2 cases). In dilated cardiomyopathy in 3 out of 5 cases an additional protein spot was found in the myocardial myosin light chain 1 area.
Maximum entropy image reconstruction of DNA sequencing gel autoradiographs
Authors: Elder John K. 
Published: May 2005

AbstractDNA sequencing gel autoradiographs become increasingly difficult to read as one moves up the gel, due to crowding and overlapping of the bands. Maximum entropy image reconstructions of the autoradiograph improve the ease with which crowded sequence data can be read, and extend the region of the gel in which reading is possible. Superior reconstructions are obtained by using nonuniform models based on the initial reconstructions.
Scale‐up of free‐flow electrophoresis: II. Purification of alcohol dehydrogenase from a crude yeast extract by field step electrophoresis and combined field step‐zone electrophoresis
Authors: Hoffstetter‐Kuhn Sabrina , Wagner Horst 
Published: May 2005

AbstractResults of the purification of alcohol dehydrogenase (ADH) by field step electrophoresis and combined field step‐zone electrophoresis are presented. In field step electrophoresis, optimization of voltage, residence time and pH of the sample solution led to a maximal purification factor of 2.8 and a yield of 89% ADH. The limit of loading capacity was reached at a protein concentration of the sample solution of approximately 4 g/L, allowing a maximal throughput of 1.14 g/h with a yield of 86% and a 2.8‐fold purification in the Elphor VaP 22 apparatus. With a production scale apparatus a throughput of 2.07 g/h without any loss of separation quality could be achieved. By introducing the sample solution into the separation chamber through 3 inlets, simultaneously, the throughput was increased to 3.2 g/h with a purification factor of 2.7 and a yield of 82% ADH. For the combined field step‐zone electrophoresis method a maximum purification factor of 3.6 and a yield of 80% ADH were achieved. The loading capacity was limited to a 4.13 g/L protein concentration of the sample solution, resulting in a throughput of 440 mg/h. Injecting the sample solution simultaneously into 3 inlets resulted in a maximum throughput of 1.92 g/h with 3.1‐fold purification and a yield of 80% ADH. Zone electrophoresis, field step electrophoresis and a combination of both are compared with respect to resolution, throughput and the application potential in a protein purification scheme. A scale‐up to 3 g/h is possible in zone electrophoresis and field step electrophoresis. The highest yields and the best purification were achieved in zone electrophoresis with a purification factor of 4.7 while on field step electrophoresis this factor was only 2.8. The combination of both technique was less efficacious with respect to throughput, yield and purification factor. Due to the different sample concentrations used, zone electrophoresis and field step electrophoresis are complementary in a purification scheme.
Interlaboratory comparative study of the numerical analysis of one‐dimensional sodium dodecyl sulphate‐polyacrylamide gel electrophoretic protein patterns of Campylobacter strains
Authors: Costas Menelaos , Pot Bruno , Vandamme Peter , Kersters Karel , Owen Robert J. , Hill Leslie R. 
Published: May 2005

AbstractTwenty‐nine bacterial strains of the genus Campylobacter were examined independently at two collaborating institutes, the National Collection of Type Cultures, London and the Laboratorium voor Microbiologie Rijksuniversiteit Gent. The onedimensional polyacrylamide gel electrophoretic protein patterns of the strains were analysed using computerised numerical methods which employed a correlation coefficient and a clustering algorithm. The electrophoretic methods used at the two institutes included both major differences such as gel composition and running conditions and minor differences in buffer composition. Although the algorithm on which similarity and clustering were computed were the same, the detailed treatment of scan patterns differed. The resulting protein patterns in the gels differed markedly in appearance but after numerical analysis the two systems were equally effecive in their ability to speciate the strains. There were, however, differences in the relationships between the species defined at the two institutes and these were at least partly due to the different background subtraction methods employed. In conclusion, the portability and reproducibility of the two systems for identification was demonstrated but for definitive classification further standardization may be required.
Polyacrylamide gel electrophoretic studies on the selfassociation of crotamine: Characterization and molecular dimension of n‐mer species
Authors: Hampe Oscar G. , Junqueira Nelson O. , Vozári‐Hampe Magdolna M. 
Published: May 2005

AbstractPolyacrylamide gel electrophoresis (PAGE) at pH 4.4 was used to study the concentration dependence of absolute mobility of crotamine. Within amounts ranging from 5–65 μg the toxin appeared in at least three n‐mer species which were characterized by their geometrical mean radius R̄ and molecular weight Mr estimation. The R̄ and Mr values of crotamine bands were obtained from equations described in the literature and by using standard polypeptides and proteins submitted to the same experimental conditions. When amounts of up to 20 μg were assayed by PAGE the bands had a monomer molecular weight value of 4650 and R̄ was 1.08 nm. From 20–35 μg the toxin migrated as dimer (Mr 10 000) with an R̄ value of 1.42 nm. However, amounts higher than 35 μg crotamine were mostly resolved in a „two‐band”︁ pattern with R̄ and Mr values corresponding to higher associated species.
Isoelectric focusing of human parotid salivary proteins in hybrid carrier ampholyte‐immobilized pH gradient polyacrylamide gels
Authors: Khoo Kong Soo , Beeley Josie A. 
Published: May 2005

AbstractIsoelectric focusing of human salivary proteins with carrier ampholyte‐isoelectric focusing systems requires prior desalting and concentration of samples, a procedure which is time‐consuming and requires relatively large volumes of samples. By contrast, immobilized pH gradient gels are more tolerant to salt loads. Thus pretreatment of samples consists only of centrifugation prior to isoelectric focusing. If larger loads (> 50 μg) are required, the samples may be concentrated by lyophilization and reconstitution in a smaller volume of water or by dialysis against 30% w/v polyethylene glycol. Immobilized pH gradient polyacrylamide gels (incorporating a hybrid carrier ampholyte system) of two pH ranges (pH 4–9 and pH 3.5–5.0) have been used to separate the proteins in human parotid saliva. The effects of urea on focused patterns were studied; in pH 4–9 gels it gave improved resolution of protein bands, whereas in pH 3.5–5.0 gels it prevented protein precipitation. The salivary proteins were then visualized by staining with Coomassie Brilliant Blue G‐250 or a silver procedure. Using the latter, 25–30 well‐resolved bands were formed on a pH 4–9 gel loaded with 20 μg of proteins. The method offers considerable advantages compared with carrier ampholyte‐isoelectric focusing.
Estimation of biotinylated lectin by isoelectric focusing
Authors: Harada Hiroshi , Kondo Maki , Yamaguchi Takashi 
Published: May 2005

AbstractConcanavalin A (Con A) was biotinylated to various degrees using N‐biotinyl‐ω‐aminocaproic‐acid‐N‐hydroxy succinimide ester as the biotinylation reagent, and then analyzed by isoelectric focusing using PhastGel IEF 3–9. The isoelectric points of biotinylated ConAs were found to decrease with increasing concentration of the biotinylation reagent. Analysis by isoelectric focusing followed by dot blotting clearly indicated that the biotinylated ConA with an isoelectric point lower than that of the original ConA by 2.2 ± 0.6 had the strongest binding activity for ovalbumin.