bluuebunny/crossref_metadata_2025 huggingface dataset:
Modified diffusion blotting for rapid and efficient protein transfer with PhastSystem
Authors: Braun Wolfgang , Abraham Rolf
Published: May 2005

An easy‐to‐perform and efficient blotting method was developed for the transfer of proteins separated in PhastGel media. The method was evaluated by comparing the blotting results of low molecular weight marker proteins at different times. 14C‐Labelled proteins were used for the assessment of the transfer efficiency. Highly efficient transfer was achieved within 1 h of blotting and the proteins were almost completely eluted from the gel in 2 h. The gel remained on its solid plastic backing, which is not the case when using an electrophoretic transfer method. As little as 1.25 ng of electrophoretically separated protein were enzymatically detected on the blot when applying blot‐biotinylation. The transfer was performed at low temperature (4°C), which – in contrast to thermoblotting – additionally offers the chance to blot thermolabile proteins. These superior features, together with the method's versatility and low cost, mark an interesting alternative to the previously recommended blotting procedures after PhastSystem electrophoresis.
Preparative separation of poliovirus structural polypeptides by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, copper staining and electroelution, and induction of monospecific antisera
Authors: Tessmer Uwe , Dernick Rudolf
Published: May 2005

Viral polypeptides were prepared by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) followed by copper staining and electroelution from gel slices. Poliovirus capsid polypeptide VP 1 isolated by this procedure induced monospecific antibodies in rabbits, i. e., antisera reacting only with the homologous polypeptide. Our results demonstrate the applicability of the described copper staining method as a rapid visualization step for preparing viral proteins after SDS‐PAGE.
Migration properties of circular DNAs using orthogonal‐field‐alternation gel electrophoresis
Authors: Hightower Robin C. , Santi Daniel V.
Published: May 2005

The migration of a series of supercoiled plasmids ranging in size from 4 to 91 kilobases (kb) has been analyzed by orthogonal‐field‐alternation gel electrophoresis (OFAGE). These circular DNAs enter OFAGE gels and are resolved over the same region of the gel as linear DNAs from 260 to 2200 kb. Furthermore, a distinct triphasic migration pattern was observed for the supercoiled DNAs. The migration of plasmids between 6 and 20, and 60 and 91 kb is inversely proportional to size, whereas the mobilities of plasmids between 20 and 60 kb increase with size. Unlike linear DNA molecules, the relative mobilities of these plasmids are constant over a broad range of pulse times, from 10 to 120 s. Electrophoresis of supercoiled, relaxed, and nicked open circular forms as well as topoisomers of small plasmids shows that the extent of supercoiling has a dramatic effect on plasmid migration on OFAGE. Several practical applications for exploiting the different migration properties of circular and linear DNA molecules on OFAGE are presented.
Temperature‐Gradient gel electrophoresis of nucleic acids: Analysis of conformational transitions, sequence variations, and protein‐nucleic acid interactions
Authors: Riesner Detlev , Steger Gerhard , Zimmat Rolf , Owens Robert A. , Wagenhöfer Manfred , Hillen Wolfgang , Vollbach Silke , Henco Karsten
Published: May 2005

Temperature‐gradient gel electrophoresis (TGGE) is applied to analyze conformational transitions and sequence variations of nucleic acids and protein‐nucleic acid interactions. A linear and highly reproducible temperature‐gradient is established perpendicular or parallel to the direction of the electrophoresis. The instrument consists of an electrically insulated metal plate, which is heated at one edge and cooled at the other edge by two thermostating baths and is used as an ancillary device for commercial horizontal gel electrophoresis instruments. Biopolymers are separated in TGGE according to size, shape and thermal stability of their conformational transitions. If the temperature‐gradient is established perpendicular to the electrophoresis, monomolecular conformational transitions of nucleic acids show up as continuous transition curves; strand‐separation leads to discontinuous transitions. In the studies on viroid RNA it was shown that natural circular viroid RNA undergoes one highly cooperative transition detected by TGGE as a drastic retardation in mobility. Oligomeric replication intermediates of viroids exhibit coexisting structures which could not be detected by any other technique. Double‐stranded satellite RNA from cucumber mosaic virus is a mixture of sequence variants, all of which have the identical length of 335 nucleotides. In TGGE six different strains were resolved. Sequence variants of viroids were analyzed by hybridizing viroid RNA to (–)strand viroid RNA transcripts from viroid cDNA clones. Sequence variations lead to mismatches in the double strands and thereby to a shift of the transition curve to lower temperature. Mutations in plasmids, particularly in cloned inserts, were detected by mixing plasmids of two different clones, linearizing, denaturing, renaturing, and searching for shifts in the transition curves, which are generated by mismatch‐formation during the renaturation of (+)‐ and (−)strands from different clones. Examples are given for different viroid clones and HIV‐clones from one and the same patient. In another example, clones with point mutations from site‐directed mutagenesis are analyzed and selected by TGGE. TGGE is also applied to study the effect of amino acid exchanges in the Tet repressor from E. coli on the thermal stability of the represser and on the mode of binding of the repressor to the operator DNA. The results are discussed under the aspect that TGGE may be applied as routine analytical laboratory procedure.
Demonstration of cell‐ricin interaction by electrophoretic light scattering
Authors: Hachemi Bahman Hakim , Betrencourt Claudine , Volochine Boris , Frénoy Jean‐Pierre , Chraibi Zakaria , Alfsen Annette , Lavialle FrançOise
Published: May 2005

Electrophoretic light scattering has been used to investigate the interaction of ricin, a vegetal toxin, with cells. This technique allowed measurements in the presence of free ligand and proved particularly useful for the study of a system with low affinity. The electrophoretic mobility of erythrocytes and oligodendrocytes was found equal to 2.08 × 10−8 and 2.35 × 10−8m2s−1 V−1, respectively. Upon ricin binding, these values decreased significantly. This change was related to the saturation of the binding sites. The specificity of the interaction was demonstrated by conducting the experiments in the presence of lactose. This specific inhibitor fully prevented the ricin‐cell interaction.
Fast isoelectric focusing of milk proteins on small ultrathin polyacrylamide gels containing urea
Authors: Moio Luigi , Luccia Aldo Di , Addeo Francesco
Published: May 2005

The 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

Two 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

Probing 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

Most 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

A 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

A 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

The 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

Acidic 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

Three 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

Isoelectric 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

Human 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

An 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

A 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

Human 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

DNA 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

Results 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

Twenty‐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

Polyacrylamide 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

Isoelectric 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

Concanavalin 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.
Protein blotting followed by microsequencing
Authors: Choli Theeodora , Wittmann‐Liebold Brigitte
Published: May 2005

The use of new membranes such as activated or derivatized glass fibers as well as synthetic membranes, which are compatible with the hazardous sequencing reagents, are described. Precautions to be taken in order to prevent N‐terminal block age of the proteins during electrophoresis and blotting are described, as well as the conditions for protein detection after blotting and protein treatment for in situ amino acid analysis, fragmentation and microsequencing. For a number of standard proteins and bacterial ribosomal proteins microsequence analysis is reported for two commercially available sequencers (Applied Biosystems and Knauer).
A gamma‐ray detector for capillary zone electrophoresis and its use in the analysis of some radiopharmaceuticals
Authors: Altria K. D. , Simpson Colin F. , Bharij A. K. , Theobald A. E.
Published: May 2005

The construction and evaluation of a gamma‐ray detector for capillary zone electrophoresis is described. The detector was shown to have a linear response from the limit of detection 10 to 500 Bqcm−3 corresponding to 5.1 x 10−17 to 2.55 x 10−15 gcm−3 43Tc99m. The application of the detector for the analysis of some radiopharmaceuticals is presented.
Applications of capillary electrophoresis for industrial analysis
Authors: Stover Frederick S.
Published: May 2005

Capillary electrophoresis techniques, particularly isotachophoresis and zone electrophoresis, are useful for the determination of various analytes in a wide range of sample matrices. This paper reports applications of capillary electrophoresis developed for detergent, food additive, herbicide, animal nutrition and biotechnology samples. Major advantages of capillary electrophoresis include versatility and speed of method development. Options for small ion analysis using modern, fused silica capillary instruments are discussed.
Enantiomer resolution by using capillary zone electrophoresis: Resolution of racemic tryptophan and determination of the enantiomer composition of commercial pharmaceutical epinephrine
Authors: Fanali Salvatore , Boček Petr
Published: May 2005

The enantiomers of D‐ and L‐tryptophan were separated by capillary electrophoresis, using α‐cyclodextrin as a chiral active component in the background electrolyte. The separation of (‐) and (+) epinephrine was achieved by supplementing the background electrolyte with Heptakis (2,6‐di‐O‐methyl‐β‐cyclodextrin). As a practical application of the method, the quantitative analysis of (‐)and(+)enantiomers in commercial pharmaceutical solutions of adrenaline is shown.
Capillary electrophoretic determination of S‐carboxymethyl‐L‐cysteine and its major metabolites in human urine: Feasibility investigation using on‐column detection of non‐derivatized solutes in capillaries with minimal electroosmosis
Authors: Tanaka Yoshio , Thormann Wolfgang
Published: May 2005

The capillary electrophoretic analysis of S‐carboxymethyl‐L‐cysteine and some of its metabolites in human urine is reported using (i) on‐column detection of underivatized solutes, (ii) minimal sample pretreatment and (iii) capillary columns with minimized electroosmosis. Experimental results obtained with two apparatus, the HPE‐100 (Bio‐Rad), featuring coated fused silica capillaries of 25 μm i.d., and with the Tachophor 2127 (LKB), having Teflon capillaries of 500 μm i.d. are discussed. Drug concentrations down to 0.2 mg/mL on capillary isotachophoresis and 0.03 mg/mL on capillary zone electrophoresis can be monitored with these instruments.
The use of metalion‐supplemented buffers to enhance the resolution of peptides in capillary zone electrophoresis
Authors: Mosher Richard A.
Published: May 2005

The potential of metal ion‐containing buffers to enhance the resolution of peptides in capillary zone electrophoresis was evaluted. The impact of adding Cu(II) and Zn(II) salts to electrophoresis buffers is shown to affect the migrational behavior of several dipeptides containing histidine. Interaction with a metal ion differentially decreases the electrophoretic mobilities of peptides which comigrate in the absence of metal ions, thus causing their separation. This effect is obtained at low pH where the large net charge on the samples yields short analysis times. The dependence of the resolution on Zn(II) concentration is presented for two different samples. The influence of the background buffer is discussed.
The effects of electroosmosis on the structure of isotachophoresis boundaries
Authors: Saville Dudley A.
Published: May 2005

The influence of convection on the structure of an isotachophoretic boundary is investigated using an asymptotic method to simplify the conservation equations. To illustrate the methodology it is applied to a simple strong electrolyte system consisting of three ions and the relevant equations solved numerically. The technique, however, is of general applicability and can be useful in exploring the influence of various flow processes on analytical and preparative electrophoretic separation techniques in addition to isotachophoresis.
Comparative studies of recycling isoelectric focusing and continuous flow electrophoresis: Separation of proteins with minor charge differences
Authors: Knisley Keith A. , Rodkey L. Scott
Published: May 2005

Continous flow zone electrophoresis (CFE) and recycling isoelectric focusing (RIEF) are two of the alternative formats for fluid phase preparative isolation of biological products in liquid separation media. The McDonnell Douglas CFE system has been used for both ground‐based and microgravity separations. The ground‐based McDonnell Douglas CFE and RIEF were compared for the ability to reslove mixtures of proteins with known charge differences. Mixtures of (1) cytochrome c, myoglobin, and ovalbumin or (2) beta‐lactoglobulin and ovalbumin were used to evaluate the resolving capabilities of CFE and RIEF. Following separation, fractions were analyzed by determining absorbance at 280 nm and by analytical isoelectric focusing (IEF) using Coomassie Brilliant Blue or silver staining to detect focused proteins. Both CFE and RIEF apparently separated the components of both mixtures into individual peaks, separated by fractions which contained little or no detectable protein. Coomassie‐stained analytical IEF gels supported this finding. However, when separated proteins were analyzed by silver staining of the analytical gels, the separation of ovalbumin from beta‐lactoglobulin by CFE was not complete. Ovalbumin was free ofbeta‐lactoglobulin but beta‐lactoglobulin was contaminated by trace amounts of ovalbumin. RIEF clearly separated each protein with no detectable contamination. These data demonstrate the superiority of RIEF over CFE for resolution of protein mixtures having only minor charge differences. RIEF may be more efficient due to the documented electrodissociation of noncovalent protein:protein complexes which occurs during RIEF separations.
Correlation of capillary zone electrophoresis with continuous free‐flow zone electrophoresis: Application to the analysis and purification of synthetic growth hormone releasing peptide
Authors: Prusik Zdeněk , Kašička Václav , Mudra Petr , Štěpánek Jiří , Smékal Otto , Hlaváček Jan
Published: May 2005

Two carrier‐free electrophoretic separation methods, capillary zone electrophoresis (CZE) and continuous free‐flow zone electrophoresis (FFZE), have been applied to both microanalysis at the nanogram level and preparative fractionation, with a throughput of 30 mg/h, of synthetic growth hormone releasing peptide (GHRP). A crude product of GHRP, a hexapeptide with the sequence His‐D‐Trp‐Ala‐Trp‐D‐Phe‐Lys‐NH2, synthesized by the solid phase methodology, was desalted and analyzed by CZE. Based on the results of analytical CZE the separation was converted into a preparative purification procedure by continuous FFZE, employing the same separation medium (0.5 mol/L acetic acid, pH 2.6). The purity of peptide frcttions obtained by FFZE was reevaluated by CZE. The combination of these two techniques proved to be a valuable tool for both peptide analysis and peptide purification. A close correalation of CZE and FFZE, resulting from the fact that both methods are based on the same separation principle (zone electrophoresis) and that both are performed in a free solution of the same composition, was confirmed. However, when transforming data from CZE to FFZE, the different electroosmotic flow, temperature and electric field intensity in the capillary and in the flow‐through cell, respectively, have to be taken into account and corresponding corrections have to be made.
Separation of enzymes from Candida boidinii crude extract by continuous flow zone electrophoresis
Authors: Nath Sunil , Schütte Horst , Weber Gerhard , Hustedt Helmut , Deckwer Wolf‐Dieter
Published: May 2005

Separation of the enzymes formate dehydrogenase, formaldehyde dehydrogenase and methanol oxidase from Candida boidinii crude extract has been explored using continuous flow zone electrophoresis in the VaP‐22 and the scaled‐up VaP‐220 electrophoresis apparatus. Yields up to 95 % and purification factors between 3 and 7 were obtained, together with separation of cell debris from the enzymes. Multiple injections of sample were used to obtain a protein throughput of 46.2 mg/h in the VaP‐22. A tenfold higher throughput was achieved using the VaP‐220. Correlation of the electrophoretic mobility in continuous flow zone electrophoresis with the elution behavior in ion‐exchange chromatography confirmed the primary role of net surface charge in the separation of biological molecules. Proteins and enzymes with differences > 0.05 M elution molarities in ion‐exchange chromatography can be separated. This corresponds to a preparative scale (mg/h or g/h) separation of proteins and enzymes whose difference in apparent electrophoretic mobility is > 0.70 x 10−5cm2/(V.s).
Nonamphoteric isoelectric focusing: II. Stability of borate‐glycerol pH gradients in recycling isoelectric focusing
Authors: Todd Paul , Elsasser William
Published: May 2005

By complexing polyols with borate in recycling isoelectric focusing and by varying the ratio of polyol to borate over the useful pH range of 4.0‐6.0, it is possible to control pH. Twelve solutions of 0.1 M boric acid and varying glycerol concentration were used to vary pH in a twelve‐compartment commercial recycling isoelectric focusing (RIEF) system. Various concentrations of boric acid were tested as anolyte, and various Tris(hydroxymethylamino)methane‐borate buffer systems were tested as catholyte. Electroosmosis, hydrogen ion flow, and fluid balancing were characterized in two glycerol gradients; one was maintained at 0.06 pH/fraction and the other at 0.12 pH/fraction. In the latter case, ovalbumin (pI 4.70) migrated to the pH 4.61 and 4.72 compartments. It is concluded that the borate‐glycerol system can be adequately stabilized in RIEF for isoelectric purification of certain proteins.
Shifts of isoelectric points between cellular and secreted antibodies as revealed by isoelectric focusing and immobilized pH gradients
Authors: Wenisch Elisabeth , Reiter Susanne , Hinger Susanne , Steindl Franz , Tauer Christa , Jungbauer Alois , Katinger Hermann , Righetti Pier Giorgio
Published: May 2005

Charge microheterogeneity of monoclonal antibodies, as revealed by isoelectric focusing in carrier ampholytes, has been known for a long time. Here we demonstrate, in the case of monoclonals against the gp‐41 of the HIV‐1 virus, that this heterogeneity is already present within the cell sap of hybridoma cells during antibody synthesis. When the monoclonals are secreted extracellularly, the same isoelectric point (pI) spectrum is maintained, but there is a marked redistribution of the relative isoform abundance towards the lower pI components. This suggests in vivo processing of such forms, possibly via glycosylation or deamidation. The secreted antibodies are also analyzed by immobilized pH gradients (IPG), where they demonstrate an even more extensive heterogeneity, due to the marked increment in resolving power. Single bands are purified by preparative IPGs in a multicompartment electrolyzer and are shown to be stable with time. Thus, artefactual heterogeneity produced by the focusing technique is completely excluded and cellular processing is clearly established.
Effects of methanol and urea on optical resolution of phenylthiohydantion‐DL‐amino acids by micellar electrokinetic chromatography with sodium N‐dodecanoyl‐L‐valinate
Authors: Otsuka Koji , Terabe Shigeru
Published: May 2005

Effects of methanol and urea on enantiomeric resolution of phenylthiohydantoin (PTH)‐DL‐amino acids by micellar electrokinetic chromatography with sodium N‐dodecanoyl‐L‐valinate (SDVal) were investigated. PTH‐DL‐Met that could not be optically resolved with an SDVal solution alone was resolved by using an SDVal/methanol solution. Four PTH‐DL‐amino acids were separated from each other and each pair of enantiomers was also optically resolved, although seriously tailed peaks were observed for each solute. The addition of urea to SDVal/methanol solutions improved these peak shapes.
Imaging as a tool for improving length and accuracy of sequence analysis in automated fluorescence‐based DNA sequencing
Authors: Sanders Jane Z. , Petterson A. Albert , Hughes Peter J. , Connell Charles R. , Raff Malcolm , Menchen Steven , Hood Leroy E. , Teplow David B.
Published: May 2005

A new method of signal analysis for automated fluorescence‐based DNA sequencing is presented. Signal resolution is a limiting factor in obtaining accurate sequence information beyond 400–450 nucleotides per gel lane. We have developed a computer program for the imaging of DNA bands in sequencing gels. The image analysis shows that distortions in the shapes of the bands decrease resolution of peaks observed in the standard data plots. Reconstruction of the undistorted band shape prior to signal analysis substantially improves the resolution of peaks and may improve the accuracy and length of the contiguous sequence read. Image analysis identified other factors limiting the accuracy and length of automated DNA sequence analysis and provided a tool for evaluating various remedies. Our techniques should also be applicable in other systems, for example, in gel electrophoresis of proteins and DNA restriction fragments, and in scranning densitometry.
Analysis of one‐dimensional gels and two‐dimensional Serwer‐type gels on the basis of the extended Ogston model using personal computers
Authors: Tietz Dietmar
Published: May 2005

This report

Preliminary reports of this study were presented at the American Electrophoresis Society Meeting, Washington DC, USA, July 6–11, 1989; at the NIH/ADAMHA‐Industry Collaboration Forum, Bethesda, Maryland, USA, October 3, 1989; at the Electrophoresis Forum '89, Munich, Germany, October 23–25, 1989; and at Frontiers in Bioprocessing II, Boulder, Colorado USA, June 17–21, 1990.

presents the stand‐alone computer application ELPHOFIT, a software package for the analysis of gel electrophoretic data based on Ferguson plots. Either conventional one‐dimensional gels or two‐dimensional agarose gels (Serwer‐type) can be evaluated. Special emphasis is on the latter gel type, which has been applied previously for the separation of DNA, intact viruses and polydisperse meningitis vaccines. ELPHOFIT is designed for Macintosh PCs and for the IBM XT, AT, PS/2 and compatibles. The program operates interactively with the user, who determines the course of evaluation. Data input is in the format of files providing values of gel electrophoretic migration distances or particle mobility (absolute or relative). Data processing involves a simultaneous least‐square curve fitting algorithm (Newton‐Gauss, Marquardt‐Levenberg) which uses equations derived from the extended Ogston model. Functions are fit to the database by adjusting their variables, representing physical parameters of the gel and the electrophoresed particle. The program output consists of tables and graphics accompanied by an explanatory text providing the following information: (i) radius and free mobility of the electrophoresed particle, (ii) fiber radius, length and volume, mean or median pore radius of the gel, (iii) linear Ferguson plots, (iv) iso‐free‐mobility/iso‐size nomogram for two‐dimensional gels, (v) confidence ellipses, (vi) required parameters or image processing program GELFIT and (vii) goodness‐of‐fit and other statistical parameters, such as standard errors, dependency values, root‐mean‐square (RMS) error and determination coefficient. Other features of the program are (i) simulation of Serwer‐type two‐dimensional electrophoresis, (ii) standardization according to size, or size and free mobility, (iii) the conversion of particle radii to molecular (or particle) weight and vice versa, (iv) interconversion of DNA size specifications, i.e. the number of base pairs and the geometric mean radii, (v) computation of gel concentration for optimal resolution of two components, (vi) option to obtain a session record, (viii) option to establish a data output file containing the information of generated graphics (IBM only) and (ix) a text editing facility, e.g., for creating data files. Graphics (Macintosh version, PICT format) and text output files (both IBM and Macintosh versions, standard ASCII format) generated by ELPHOFIT are compatible with commercially availabe software.