bluuebunny/crossref_metadata_2025 huggingface dataset:
Micellar electrokinetic capillary chromatography analysis of water‐soluble vitamins and multi‐vitamin integrators
Authors: Dinelli Giovanni , Bonetti Alessandra
Published: May 2005

Micellar electrokinetic capillary chromatography (MECC) was applied to the determination of water‐soluble vitamins (WSV) in pharmaceutical preparations. The analytical procedure employed in the WSV separation by MECC showed a satisfactory average column efficiency (195000 N) and resolution (more than 100 theoretical peaks separable). The mean reproducibility in the retention time of consecutive and day‐to‐day runs was 1.1% and 5.1%, respectively. Analyses were performed after extraction of active ingredients by solid phase extraction. Recoveries (92–103%) and relative variation coefficients (less than 5%) confirmed the potential of MECC in the field of pharmaceutical analysis.
Isolation of human serum transferrin by free‐fluid recycling electrophoresis in simple buffers
Authors: Caslavska Jitka , Thormann Wolfgang
Published: May 2005

The isolation of human serum transferrin (Tf) using recycling isotachophoresis (RITP) and recycling isoelectric focusing (RIEF) with simple buffers is described. Serum fractionation, the first step in the protocol for Tf purification, is shown to be easily performed either by RITP of filtered serum using low molecular mass spacers or by RIEF of dialyzed serum employing a binary mixture of a well‐defined buffer pair covering the pH range between 5.2 and 6.2, called RIEF‐OptiFocus. For polishing, Tf‐containing fractions are reprocessed by RITP or RIEF‐OptiFocus. Other RIEF approaches based on the use of single amino acids at high concentration and ternary amino acid mixtures are shown to constitute less effective methods for Tf isolation. With a four‐step protocol, comprising in turn RITP, RIEF‐OptiFocus, ultrafiltration and again RITP, “single‐band” purity (as assessed by two‐dimensional gel electrophoresis) is obtained. Omission of the first step (RITP) and direct processing of dialyzed serum by RIEF‐OptiFocus, ultrafiltration and RITP, is shown to provide remarkable results as well.
Analysis of the inter‐alpha‐trypsin inhibitor polymorphism in West Africa: Description of a new allele, ITI*7
Authors: Caeiro José Luis B. , Liste Isabel , Vogt Ulrike , Ribeiro Joaquim C. Teixeira
Published: May 2005

Inter‐α‐trypsin inhibitor (ITI) phenotypes were classified in the West African population of Cabo Verde by polyacrylamide gel isoelectric focusing, followed by immunofixation and silver staining. Gene frequencìes of the alleles ITI*1, ITI*2, ITI*3 and ITI*4 were calculated to be 0.532, 0.153, 0.307 and 0.002, respectively. A new rare allele, ITI*7, was found, providing evidence for further genetic variability of the ITI protein. The ITI*7 allele frequency has been determined to 0.006. The assumption that allele ITI*3 may be used to characterize populations of African origin is supported by our data.
Mixed micellar electrokinetic chromatography of corticosteroids
Authors: Bumgarner Jefferson G. , Khaledi Morteza G.
Published: May 2005

Mixed micellar electrokinetic chromatography (MMEKC) using mixtures of bile salt surfactants and/or sodium dodecyl sulfate (SDS) was employed to separate a group of corticosteroids. Resolution in electrokinetic chromatography (EKC) can be greatly enhanced by the use of mixed micellar systems due to the fact that the composition of the mixed micelles has a great influence on retention, selectivity and the size of the elution window. By combining surfactants with different structural properties, solute‐micelle interactions were manipulated in order to elicit specific separations. Various combinations of bile salts and/or SDS at different mole fractions as well as total micelle concentrations were used in order to enhance the resolution of corticosteroid separations. Large changes in retention and selectivity were observed that often resulted in frequent variations in elution order. In addition, the composition of mixed micellar systems had a great influence on the elution window in EKC, as measured by the ratio of tmc/ teo. Addition of SDS to the mixtures of bile salt micelles resulted in significant extension of the elution window and subsequently improvement in resolution. A separation of seventeen corticosteroids was achieved. Finally, MMEKC was applied in order to separate the steroidal components of a mixture of three anti‐inflammatory creams.
The use of mixed surfactants in micellar electrokinetic chromatography
Authors: Ong Chye P. , Ng Choon L. , Lee Hian K. , Li Sam F. Y.
Published: May 2005

The use of mixed surfactants for the separation of nine phenylthiohydantoin amino acids (PTH amino acids) by micellar electrokinetic chromatography (MEKC) was examined. The potential of a mixed surfactant system, consisting of cationic and anionic surfactants, for enhancing the selectivity of MEKC separations was explored. Both sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide (CTAB) in the electrophoretic medium were found to improve the separation of the PTH‐amino acids compared to the use of CTAB or SDS alone. Complete separation of the PTH amino acids was achieved using a buffer containing 2.5 mM CTAB and 40 mM SDS. The analysis was performed without the need to reverse the polarity of the electrodes, typically required if oppositely charged micelles are used. The migration behavior of the nine PTH amino acids using the mixed surfactant system was compared with MEKC in presence of SDS alone.
Micellar electrokinetic chromatographic studies in D2O based buffer solutions
Authors: Greenaway Mark , Okafo George , Manallack David , Camilleri Patrick
Published: May 2005

The addition of deuterium oxide or organic solvents to surfactant‐based electrolytes is an effective method for improving resolution in micellar electrokinetic capillary chromatography (MECC). Buffers containing phosphate, boric acid and sodium dodecyl sulphate at pH or pD 7 in the presence and absence of deuterated and non‐deuterated solvents were used to separate some hydrophobic compounds covering a range of octanol‐water partition coefficients (log P). Significant improvements in resolution of the more hydrophobic compounds were achieved by using mixtures of heavy water and deuterated methanol, instead of water and methanol. Quantitative structure‐retention relationships were also derived to provide a better understanding of the intermolecular interactions that contribute to the separation of analytes in MECC. Multiple regression analysis has shown that capacity factors were significantly related to both log P and molecular refractivity. The latter is a measure of the molecular volume of analytes and is related to their ability to undergo dispersive interactions with the micellar components.
Micellar capillary electrophoresis separation and thermo‐optical absorbance detection of products from manual peptide sequencing
Authors: Chen Min , Waldron Karen C. , Zhao Yuwei , Dovichi Norman J.
Published: May 2005

Micellar capillary electrophoresis is optimized for separation of phenylthiohydantoin (PTH) amino acids produced in manual Edman degradation reaction for protein sequencing. There are also two major side‐products produced by the Edman degradation reaction: diphenylthiourea and dimethylphenylthiourea. We report the complete separation of 19 PTH amino acids plus the two major side‐reaction products in 10 min. Capillary electrophoresis is used to identify the five residues generated by manual Edman degradation sequencing of a pentapeptide.
Micellar electrokinetic chromatography of the anti‐human immunodeficiency virus (HIV) agent michellamine B with absorption and laser‐induced fluorescence detection
Authors: Chan King C. , Majadly Fathy , McCloud Thomas G. , Muschik Gary M. , Issaq Haleem J. , Snader Kenneth M.
Published: May 2005

Micellar electrokinetic chromatography (MEKC) was applied to the separation of the anti‐HIV agents, michellamines A and B, and two other structurally related monomers found in the extract of the Ancistrocladus plants. Using buffers containing either 10 mM sodium phosphate (pH 7.0), 50 mM sodium de‐oxycholate and 10–20% acetonitrile or 5 mM sodium phosphate (pH 7.0), 20 mM sodium dodecyl sulfate and 25% acetonitrile allowed baseline separations of the four components in the mixture in less than 10 min. The MEKC methods gave sharper peaks and better resolution compared to high‐performance liquid chromatography. For MEKC separation of the plant extracts, UV absorption detection provided adequate sensitivity; however, higher sensitivity could be achieved with UV laser‐induced fluorescence detection (LIF). Using the sodium dodecyl sulfate‐containing buffer and LIF, the limit of detection for michellamine B was ∼ 2 ng/mL. The sensitivity was degraded ∼100‐fold when using the deoxycholate buffer because of high background fluorescence. Preliminary results show that MEKC with LIF is feasible for the sensitive detection of michellamine B in serum.
The human myocardial two‐dimensional gel protein database: Update 1994
Authors: Corbett Joseph M. , Wheeler Colin H. , Baker Cathy S. , Yacoub Magdi H. , Dunn Michael J.
Published: May 2005

An updated human heart protein two‐dimensional electrophoresis (2‐DE) database is presented. The database, which contains some 1388 protein spots characterised in terms ofMrand pI, has been analysed further by Western immunoblotting and protein sequencing. From a total of 103 protein spots analysed, 49 have been identified by immunoblotting and 32 have been identified by protein sequencing. A further six proteins have tentatively been assigned by comparison with the human heart 2‐DE protein database of Jungblutet al. (Electrophoresis) 1994,15, 685–607). This database is being used in studies of alterations in protein expression in the diseased and transplanted human heart.
Electric birefringence imaging of DNA in agarose electrophoresis gels: Noise limits and prolonged electric field effects
Authors: Lanan Maureen , Grossmann Daniel W. , Morris Michael D.
Published: May 2005

Electric birefringence imaging (EBI) maps stresses in agarose electrophoresis gels. Both DNA and gel alignment can be monitored using this technique. Previously, we have reported birefringence images associated with resolved small (< 48 kbp) and partially resolved large 48 kb – (2 Mbp) DNA fragments oriented with relatively short electric field pulses. In this paper, differences between images acquired using short and long (DC) voltage pulses are presented. It is shown that stress signals distribute over long distances when exposed to prolonged electric fields. Resolution also affects band shapes. Refinements in instrumental design and evaluation of limiting noise sources of the technique are also described.
An algorithm for unwarping multitrack electrophoretic gels
Authors: Glasbey Chris A. , Wright Frank
Published: May 2005

An algorithm is proposed for removing the effect of “frowns” and “smiles” (i.e. warping) from multitrack electrophoretic gels, so that the bands are horizontal and in alignment between tracks. It has been applied to a DNA sequencing autoradiograph, but could equally be applied to other types of gel separation where warping occurs. The algorithm uses a gradient filter to estimate band orientations. These are smoothed and interpolated by robust regression, and integrated to find the unwarping transform. No manual intervention is required.
Calculation of paternity probabilities from multilocus DNA profiles
Authors: Brenner Charles H. , Rittner Christian , Schneider Peter M.
Published: May 2005

We describe a procedure for evaluation of paternity evidence from multi‐locus DNA probe patterns. A computer program abstracts a “+/−” notation description from the multilocus profile and then calculates a paternity index based on observed phenotypic fragment frequencies. The biostatistical evaluation considers only bands found in the child and missing from the mother ‐ a simplified approach that is at once robust and conservative. Mutations are of course taken into account. Particular features lending objectivity to the interpretation include computer reading and matching decisions, and specific recognition and statistical compensation for ambiguities (“faint orphans”).
Towards new formulations for polyacrylamide matrices: N‐acryloylaminoethoxyethanol, a novel monomer combining high hydrophilicity with extreme hydrolytic stability
Authors: Chiari Marcella , Micheletti Claudia , Nesi Marina , Fazio Marco , Righetti Pier Giorgio
Published: May 2005

Matrices for electrokinetic separations, based on a unique class of mono‐ and disubstituted (on the amido nitrogen) acrylamides such as e.g., N‐acryloylaminoethoxyethanol (AAEE) and acrylamido‐N,N‐diethoxyethanol, offer the following advantages: (i) strong resistance to alkaline hydrolysis (most zone separations occurring at basic pH values), (ii) high hydrophilicity and (iii) greater porosity, due to the higher molecular weight of the monomers. When compared with conventional poly(acrylamide), a poly(AAEE) matrix, when subjected to mild alkaline hydrolysis (0.1 N NaOH, 70°C) appears to be 500 times more stable. Such stability is also confirmed under strong alkaline hydrolysis (1 N NaOH, 100°C) as well as under mild and strong acidic hydrolysis. Mildly hydrolyzed poly(AAEE) matrices still perform extremely well in both conventional isoelectric focusing and immobilized pH gradients, techniques which are quite sensitive to traces of acrylate in the polymer coil. Conversely, mildly hydrolyzed poly(acrylamide) matrices, when used in isoelectric focusing, generate pH gradients between pH 4 and 5, having an inflection point (pH 4.6) equivalent to the pK value of acrylic acid. This novel class of monomers shows great promise for future applications in all electrokinetic methodologies.
Differential detergent fractionation of isolated hepatocytes: Biochemical, immunochemical and two‐dimensional gel electrophoresis characterization of cytoskeletal and noncytoskeletal compartments
Authors: Ramsby Melinda L. , Makowski Gregory S. , Khairallah Edward A.
Published: May 2005

Two‐dimensional (2‐D) gel electrophoresis is often used in toxicologic and metabolic studies to assess treatment‐ or stage‐specific changes in protein synthesis, degradation or posttranslational modification. When combined with cell fractionation studies the detectability of low abundance proteins is enhanced, and changes in subcellular distribution of proteins can also be monitored. Detergent fractionation is a simpler alternative to differential pelleting, which partitions cellular constituents into functionally distinct populations while preserving cytoskeletal integrity. We defined and characterized a differential detergent fractionation (DDF) protocol to enable protein dynamics in cytoskeletal and noncytoskeletal compartments of isolated hepatocytes to be monitored simultaneously. Rat hepatocytes were maintained in suspension culture and fractionated by sequential extraction with detergentcontaining buffers (digitonin/EDTA, Triton/EDTA, Tween/deoxycholate). DDF reproducibly yielded four electrophoretically distinct fractions enriched in cytosolic, membrane‐organelle, nuclear membrane and cytoskeletal‐matrix markers, respectively. Immunoblotting with over 20 different antibodies corroborated the selectivity of fractionation and was used to characterize the distribution profiles of cytoskeletal (actin, tubulins, cytokeratins, vinculin, myosin, desmoplakins, fodrin, nuclear lamins) and noncytoskeletal proteins (heat‐shock 70 proteins, glutathione‐S‐transferase, calpains, carbamoyl phosphate synthetase, etc.), as well as to identify spots in 2‐D gels. Detergent buffers were compatible with equilibrium or nonequilibrium 2‐D gel electrophoretic analysis. Extensive 2‐D maps of acidic and basic proteins in each fraction were generated along with a tabular listing of Mr and pI. Thus, DDF reproducibly partitions hepatocytic proteins into functionally distinct cytoskeletal and noncytoskeletal compartments that are readily analyzed by 2‐D gel electrophoresis. DDF is simple, applicable to use with other cell types or culture systems and is especially useful when biomaterial is limited (i.e., clinical studies).
Two‐dimensional electrophoresis of basic proteins with equilibrium isoelectric focusing in carrier ampholyte‐pH gradients
Authors: Rabilloud Thierry
Published: May 2005

A modified procedure for the two‐dimensional electrophoretic analysis of basic polypeptides is described. This method uses isoelectric focusing with carrier ampholytes in the first dimension, and sodium dodecyl sulfate‐electrophoresis in the second dimension. Counteraction of the cathodic drift is achieved by glass tube treatment (silanization), electrolyte modification (use of weak bases and acids), protection of the catholyte from carbon dioxide, and the addition of glycerol to the gel mix. Better resolution and reproducibility are obtained than with nonequilibrium pH gradient electrophoresis, since quasi equilibrium focusing can be obtained.
Mapping small GTP‐binding proteins on high‐resolution two‐dimensional gels by a combination of GTP binding and labeling with in situ periodateoxidized GTP
Authors: Huber Lukas A. , Peter Marcus E.
Published: May 2005

We compared two approaches to identify and map small GTP‐binding proteins in combination with high‐resolution two‐dimensional (2‐D) gel electrophoresis. The first approach involved direct GTP ligand binding after a renaturing transfer onto nitrocellulose. In the second, affinity labeling with in situ periodate‐oxidized GTP was used in permeabilized cells (Peter, M. E., She, J., Huber, L. A. and Terhorst, C. Anal. Biochem. 1993, 210, 77–85). Analysis by 2‐D gel electrophoresis revealed a number of distinct intracellular small GTP‐binding proteins in Madine‐darby canine kidney strain II cells (MDCKII). Using specific antibodies the electrophoretic coordinates of rab4, rap1a/b, and rap2 were identified for native as well as for crosslinked GTPases. These methods allow the identification of small GTP‐binding proteins in total cell lysates and purified subcellular fractions, providing excellent markers throughout the course of differentiation and development.
Discovery and characterization of two novel human cancer‐related proteins using two‐dimensional gel electrophoresis
Authors: Leavitt John
Published: May 2005

Comparative examination of protein synthesis in normal and neoplastic human fibroblasts led to the discovery of two novel microfilament proteins with roles in human neoplasia. One protein, a mutant β‐actin was found to convert nontumorigenic human fibroblasts to tumorigenicity. Recently, the oncogenic potential of this mutant β‐actin was verified independently and shown to alter the metastatic phenotype of human cells in conjunction with the myc and ras oncogenes. A second protein, leukocyte plastin, was discovered to be a marker of a majority of human cancer cells of nonhemopoietic origin. A survey of SV40‐transformed human fibroblasts and human sarcoma and carcinoma cell types demonstrated that the L‐plastin gene was activated at widely varying degrees in nearly all human cancer cells. Activation of the L‐plastin gene was not detected in normal nonhemopoietic cells using sensitive reverse transcript‐polymerase chain reaction, excepting those cells that expressed estrogen and progesterone receptors which mediate activation of L‐plastin synthesis in reproductive tissues. Our most recent findings have revealed that activation of L‐plastin synthesis in neoplastic cells that cannot phosphorylate L‐plastin (e.g. those neoplastic cell types that express only trace amounts of L‐plastin) results in the coinduction of two alternative inflammatory programs of gene expression which mediate cytolytic effects on surrounding cells. This inflammatory response appears to be mediated by “inappropriate” constitutive synthesis of L‐plastin and failure of the induced cell to phosphorylate L‐plastin. Our findings suggest explanations for the novel resistance of human cells to in vitro transformation and one role of oncogene activation in cancer. As a consequence of the interplay of two‐dimensional (2‐D) gel electrophoretic analyses with other sophisticated techniques of molecular biology, the formal characterization of two fundamentally important multigene families was completed with determination of many aspects of the structure and function of these proteins and their genes. The discovery and characterization of the mutant β‐actin and L‐plastin and their relationship to the human neoplastic phenotype serve as useful models for the discovery of other important disease‐related proteins/genes using 2‐D gel electrophoresis.
Expression of tumor‐associated protein variants in chemically induced rat hepatomas and transformed rat liver cell lines determined by two‐dimensional electrophoresis
Authors: Zeindl‐Eberhart Evelyn , Jungblut Peter , Rabes Hartmut M.
Published: May 2005

High resolution two‐dimensional electrophoresis (2‐DE), using the rat liver as a model, was applied to study hepatocarcinogenesis induced by different chemical carcinogens. Several tumor‐associated protein variants were detected by 2‐DE in chemically induced rat hepatomas and transformed rat liver cell lines compared to normal rat liver tissue. Proliferation‐related protein changes and/or protein alterations due to culture conditions were corrected for by comparison with 2‐DE patterns of isolated cells from regenerating liver and short‐term cultivated liver cells. Some of the tumor‐associated variants were further characterized: (i) By peptide mapping with limited proteolysis we detected clear relationships between several variants. (ii) By studying posttranslational modifications phosphorylated and glycosylated variants could be determined. (iii) A tumor‐associated protein variant was identified by amino acid analysis and amino acid sequencing.
Studying the interaction of polyoma virus middle T antigen with cellular proteins
Authors: Krauzewicz Nina S.
Published: May 2005

The oncogenic mouse polyoma virus encodes six proteins, two of which (the large and middle T antigens), when expressed together in primary rodent cells, generate an alteration of growth patterns commonly known as cellular transformation. The transformed cells grow with an unlimited life span and when introduced into immunocompromised mice rapidly form tumours. The large T gene product confers an indefinite growth potential on primary cells; however, the middle T gene product has been identified as bringing about the changes which ultimately allow the cell to form tumours. The 55 kDa middle T antigen has been shown to associate with a number of cellular enzymes involved in regulation of growth factor signalling pathways, all of which were identified as being components of the immunocomplexes that can be isolated from transformed cells using middle T antigen specific antibodies. Two‐dimensional gels have assisted the search for less prominent species present in these complexes. These methods represents one approach to investigating associating proteins, and as such, select for those interactions that are stable under the conditions used. In order to explore the possibility that middle T antigen could form complexes with other cellular proteins given different conditions, recombinant middle T antigen was used in a series of “filter overlay” experiments.
Phosphopeptide mapping and phosphoamino acid analysis by electrophoresis and chromatography on thin‐layer cellulose plates
Authors: van der Geer Peter , Hunter Tony
Published: May 2005

Identification of protein phosphorylation sites is essential in order to evaluate the contribution of individual sites to the regulation of a particular protein by phosphorylation. Here we review a method we have developed for the identification of phosphorylation sites based on digestion of 32P‐labeled proteins with site‐specific proteases and separation of the digestion products in two dimensions on thin‐layer cellulose plates using electrophoresis in the first dimension followed by chromatography. This method is very sensitive, requiring only a few hundred 32P‐disintegrations per minute to obtain reproducible phosphopeptide maps. We also report methods for the analysis of the phosphoamino acid content of both intact phosphoproteins and individual phosphopeptides recovered from two‐dimensional separations, in which the material is subjected to partial acid hydrolysis, and the hydrolysis products are separated on thin‐layer cellulose plates by electrophoresis in one or two dimensions. Finally, we describe methods for analyzing the structure of isolated phosphopeptides by secondary digestion with site‐specific proteases, by manual Edman degradation, and by immunoprecipitation, and indicate how this information can be used in conjunction with the two‐dimensional mobility of the peptide to deduce the identity of a phosphopeptide from the known sequence of a protein.
A rapid method for detection of N‐acetylglucosaminidase‐type chitinase activity in crossed immunoelectrophoresis and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis gels using 4‐methylumbelliferyl‐N‐acetyl‐D‐glucosaminide as substrate
Authors: Chen Kun‐Shang , Lee Kuo‐Kau , Chen Hsing‐Chen
Published: May 2005

A method for the detection of N‐acetylglucosaminidase (GlcNAcase) activity has been developed by using 4‐methyl‐umbelliferyl‐N‐acetyl‐D‐glucosaminide (4‐MU‐GlcNAc) as substrate in crossed immunoelectrophoresis (CIE) and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) gels. Visualization of the reaction with a 366 nm ultra‐violet light is possible in approximately 30 min. The method is fast and sensitive in comparison with previous methods. The same band as in SDS‐PAGE, showing both GlcNAcase and chitinase activity, was found in the present study; we therefore conclude that this method is also useful in a GlcNAcase‐type chitinase assay.
The homology of a novel polypeptide with stress protein characteristics in embryonic mice brain and in the hypothalamus of caloric restricted rats as determined by ultramicro western blotting
Authors: Pipkin James L. , Hinson William G. , Lyn‐Cook Lascelles E. , La Borde James , Anson Jeanne F. , Feuers Ritchie J. , Duffy Peter H. , Hart Ronald W. , Casciano Daniel A.
Published: May 2005

A novel protein (p34) was observed in polyacrylamide gel fluorographs of gestation day 13 embryonic mouse brain following retinoic acid dosing of dams. Another p34 polypeptide with identical gel migratory characteristics was seen in the hypothalamus of old caloric restricted rats after “food deprivation stress”. Western blotting, employing an ultramicro trans‐blot cell developed in our laboratory, detected identical immunochemical determinants between these proteins, verifying their homology. Peptide mapping and Western blotting further validated the uniqueness of p34 compared with other stress proteins including heme oxygenase.
Alzheimer brain proteins investigated by two‐dimensional gel electrophoresis with immobilized pH gradients in the first dimension
Authors: Mattila Kari M. , Frey Harry
Published: May 2005

Two‐dimensional (2‐D) gel electrophoresis with immobilized pH gradient 4–8 in the first dimension was applied in the analysis of Alzheimer's disease brain proteins. The silver‐stained 2‐D maps of extracts from the frontal cerebral cortex were examined. About 800 and 550 protein spots could be observed on the electrophoretograms from the total and buffer‐soluble fractions, respectively. In comparing the gels, four protein spots could be seen in all Alzheimer 2‐D patterns which were either undetectable (one spot) or significantly weaker (two spots) or stronger (one spot) in density than those from the controls.
Pulsed field separation of large supercoiled and open‐circular DNAs and its application to bacterial artificial chromosome cloning
Authors: Wang Min , Lai Eric
Published: May 2005

We have studied the separation of large (80–300 kbp) supercoiled (SC) DNA in conventional agarose gel electrophoresis, field inversion gel electrophoresis (FIGE) and pulsed field gel electrophoresis (PFGE). DNA migration was measured under a variety of electrophoretic conditions including different switch times, temperatures, agarose concentrations, and voltage gradients. The migration of SC DNA was found to be inversely proportional to its molecular weight in the three electrophoresis systems tested. In conventional agarose electrophoresis, voltage gradient was found to be the determining parameter in the separation of SC DNA. Unlike large linear DNAs, the migration of SC DNA was found to be independent of switch time in PFGE and FIGE. Broad DNA bands were observed in prolonged FIGE runs. In addition, we have also studied the migration of open‐circular (OC) DNA (80 and 100 kbp) in pulsed field gel electrophoresis. Eighty kbp OC DNA can migrate into agarose gels under certain pulsed field conditions whereas 100 kbp OC DNA was trapped at the wells. Based on electrophoretic conditions described in this report, we can determine the size of bacterial artificial chromosome (BAC) clones without restriction enzyme digestion and have enriched the percentage of larger size clones in BAC cloning.
Single‐strand conformation polymorphism analysis by perpendicular temperature‐gradient gel electrophoresis
Authors: Sugano Kokichi , Fukayama Noriko , Ohkura Hisanao , Shimosato Yukio , Yamada Yasushi , Inoue Tamotsu , Sekiya Takao , Hayashi Kenshi
Published: May 2005

Using a newly developed temperature‐gradient gel electrophoresis apparatus, mutations in the K‐ras oncogene and p53 tumor supressor gene were analyzed for single‐strand conformation polymorphism (SSCP). The mobilities of single‐stranded DNAs, carrying various mutations, change – depending on the gel temperature during electrophoresis. Therefore, temperature‐gradient single‐strand conformation polymorphism (TG‐SSCP) analysis can provide useful information concerning the optimum temperature for SSCP and may also be used to screen for various mutations or polymorphisms in a single electrophoretic run.
Protein concentration by precipitation with pyrogallol red prior to electrophoresis
Authors: Marshall Thomas , Abbott Nicola J. , Fox Peter , Williams Katherine M.
Published: May 2005

The pyrogallol red protein assay (Clinical Chemistry 1986, 32, 1551–1554) is based upon formation of a blue protein‐dye complex in the presence of molybdate under acidic conditions. However, centrifugation of the assay mixture results in loss of color yield and precipitation of the protein‐dye complex which can be recovered and resolubilized to achieve protein concentration prior to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The method has been evaluated relative to trichloroacetic acid (TCA) precipitation for recovery and electrophoresis of commercial protein and peptide molecular weight markers. Precipitation with pyrogallol red‐molybdate (PRM) gives better and more uniform recovery of both proteins and peptides as compared to TCA. The lower limit of PRM precipitation is similar to TCA and corresponds to 1 μg protein per mL assay mixture. This is equivalent to 100 μL of 10 μg/mL protein using the standard protein assay or 1 μg/mL protein using a modified assay incorporating a fivefold concentrate of the dye reagent. Application of the method is demonstrated by concentration of urinary proteins. The method is simple and economic and useful for conserving trace amounts of precious sample as it allows recovery of protein for electrophoresis following protein assay.
Separation of arginase isoforms by capillary zone electrophoresis and isoelectric focusing in density gradient column
Authors: Pedrosa Mercedes M. , Legaz Maria Estrella
Published: May 2005

Four major arginase isoforms, I, II, III and IV, have been detected in Evernia prunastri thallus. They differ in terms of both physical and biochemical properties. The isoelectric point (pI) of these proteins has been determined by both isoelectric focusing in density gradient column and high‐performance capillary electrophoresis (HPCE). Isoelectric focusing revealed charge microheterogeneity for isoforms II and IV whereas arginases I and II had the same pI value of 5.8. HPCE separation confirmed this charge microheterogeneity for isoform IV but not for isoform III, and provided evidence of microheterogneity for isoforms I and II. The effect of various electrolyte buffers and running conditions on the HPCE separation of arginase isoform were investigated. Addition of 0.5 mM spermidine (SPD) to the running buffer reduced the electroosmotic flow (EOF) and permitted discriminating between the native proteins and protein fragments.
Simple and sensitive laser‐induced fluorescence detection for capillary electrophoresis and its application to protein separation
Authors: Lim Heung Bin , Lee Jong Jin , Lee Kong‐Joo
Published: May 2005

A low‐cost and highly sensitive laser‐induced fluorescence detector for on‐column detection systems was constructed and its applications were demonstrated in protein separation by capillary electrophoresis. The limit of detection (LOD) at a signal‐to‐noise ratio of 2 for Lissamine 20 was about 1.4 × 10−21 mole at 10 nL sample injection and the relative standard deviation (RSD) for 1.5 × 10−11 M of Lissamine 20 solution was about 15%. These values are comparable to or even better than those reported using a similar on‐column detection system. This sensitive LIF detection was applied for the study of tryptic digestion of insulin and for urinary protein profiling.
Indirect DNA/gene diagnoses via electrophoresis – an obsolete principle?
Authors: Epplen Jörg T. , Buitkamp Johannes , Epplen Cornelia , Mäueler Winfried , Rieß Olaf
Published: May 2005

In principle, gene defects can be investigated directly or indirectly via informative polymorphisms in their vicinity. But because many defects are not yet defined molecularly, these inherited diseases can only be diagnosed indirectly via analysis of informative family situations. Since (multiple) mutation analyses, e.g. via DNA sequencing, are time‐consuming and expensive, indirect analysis may still be performed initially – particularly in diseases caused by heterogenous mutations. We focus on diagnoses of neurological and (auto)immune diseases by polymerase chain reaction and separation of the DNA fragments via gel electrophoreses. Even after gene defects have been identified, indirect analysis might be necessary, for example in Huntington's chorea. Although this genetic defect has been characterized as a trinucleotide disease, indirect DNA diagnosis is still performed in particular cases for psychological reasons. The causes of autoimmune diseases are multifactorial and the inheritance is complex, involving several genes. Genome‐wide screening programs may involve indirect approaches via analyses of polymorphic microsatellites. Large parts of the immunological genome can be covered when 20 or more genes are investigated simultaneously. Thus the genetic bases of autoimmune diseases are disclosed. Microsatellites themselves could have a biological meaning. We therefore discuss also DNA/protein interactions for simple tandem repeats, the major targets for indirect gene diagnoses. Only indirect evidence exists that certain simple repeats influence genomic (in)stability. Taken together, indirect gene diagnoses supplement direct approaches in a variety of different purposes and in combination with standard electrophoresis techniques.
Commercial automated gel electrophoresis apparatus: Application to DNA, band dispersion, nonlinear Ferguson curves, and isolation
Authors: Chrambach Andreas , Yarmola Elena , Zakharov Sergey F. , Garner Mark M.
Published: May 2005

Recently available commercial automated gel electrophoresis apparatus with intermittent scanning of fluorescently labeled gel patterns (the HPGE‐1000 apparatus of LabIntelligence, Menlo Park CA) was tested with regard to (i) its applicability to DNA in its native conformation, (ii) its ability to recognize the correct number of components, (iii) its capability to evaluate the width and shape of bands detected during electrophoresis, (iv) its ability to yield nonlinear Ferguson plots in a labor‐saving fashion, and (v) its preparative potential. Ethidium homodimer (EtD) DNA (bp) ratios were systematically varied and the mobility of DNA fragments labeled at each ratio was measured in order to find a ratio which provided an unaltered mobility and presumably therefore an unaltered conformation of the fragment. That ratio was found to be 1/40 EtD/ DNA (bp) or less. With such weak labeling of DNA, a representative fragment of 527 bp length requires a minimum load of 200 ng and a 2 μg load for a full‐scale peak height. Using the baseline automatically selected by the software of the apparatus, the band areas of the 17 components of a DNA digest were consistently evaluated by the software, as evidenced by the proportionality between DNA length and area. The areas of the separated bands of DNA fragments of 1857 and 121 bp length were found to be constant with time of electrophoresis. The dispersion coefficient was found to decrease with agarose concentration in electrophoresis at 1 V/cm; however, at higher field strength, the band width of the 1857 bp fragment was surprisingly found to increase with gel concentration, presumably due to stretching. Electrophoretic band dispersion was not found to be due to diffusion since zone spreading in the absence of the electric field under otherwise identical conditions does not proceed measurably. For the discontinuous buffer system of Wiltfang et al. (Electrophoresis 1993, 12, 352–366) the resolving power, in terms of theoretical plate equivalents, was approximately threefold higher compared to that in the Trisborate‐EDTA (TBE) buffer under otherwise identical conditions. Two electrophoretic runs of the apparatus with different gel concentrations in its eight channels suffice to define nonlinear Ferguson curves, provided that the free mobility is measured by capillary electrophoresis. The commercial automated apparatus excels as a preparative device by allowing one to monitor the qualitative and quantitative success of electroelution.
Biotinylation: A nonradioactive method for the identification of cell surface antigens in immunoprecipitates
Authors: Flesch Brigitte , Ntambi Elisabeth , Neppert Jürgen
Published: May 2005

A nonradioactive method was employed to detect different cell membrane antigens on human polymorphonuclear granulocytes, monocytes and platelets. We compared the reactivity of one monoclonal antibody, N1III10, assumed to be FcγRII‐specific by functional assays, with other well‐characterized monoclonal antibodies and human sera. Intact cells were incubated with biotin N‐hydroxysulfosuccinimide ester which preferentially reacts with lysine residues in polypeptides. Biotin‐labeled cells were lysed and the antigen was isolated from the cell lysate by immunoprecipitation with the antibody bound to Protein A‐Sepharose. The precipitates were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane, and visualized by a streptavidin‐alkaline phosphatase system with a suitable substrate. Using this biotin‐labeling system we could show that N1III10 detects a 40 kDa antigen on monocytes and platelets, comparable to that expected of FcγRII monoclonal antibodies.
Separation and quantitation of reverse transcriptase polymerase chain reaction fragments of basic fibroblast growth factor by capillary electrophoresis in polymer networks
Authors: Gelfi Cecilia , Leoncini Fiorella , Righetti Pier Giorgio , Cremonesi Laura , di Blasio Anna Maria , Carniti Cristiana , Vignali Mario
Published: May 2005

In human ovarian carcinomas (epithelial and endometrial tumors) the presence of mRNA coding for the basic fibroblast growth factor (bFGF) was previously demonstrated by reverse transcription‐polymerase chain reaction (PCR). For quantitation purposes, competitive PCR is adopted, using as a competing fragment a sequence of a highly homologous bovine bFGF. However, separation and detection of the PCR products by slab gel electrophoresis and ethidium bromide staining gives poor quantitative data due to the nonstoichiometric binding of the dye. Thus, the only possible quantitation that can be obtained is via autoradiography with 32P‐labeled primers. We report here a capillary electrophoresis protocol, in 6% linear, liquid polyacrylamide as a sieving system, able to fully resolve and quantify the undigested (354 bp) bovine and the digested (295 bp and 59 bp) human fragments, with peak ratios in good agreement with the autoradiographic data.
A novel strategy to identify maternal and paternal inheritance in the mouse
Authors: Römer Irmgard , Jungblut Peter , Reik Wolf , Otto Albrecht , Klose Joachim
Published: May 2005

A novel strategy for identifying proteins which reveal maternal or paternal inheritance in the mouse is presented. Using two‐dimensional electrophoresis we investigated protein expression patterns of adult liver and different embryonic and extraembryonic tissue in C57BL/6Crl and in DBA/2Crl mice, as well as in their reciprocal hybrids. We found three groups of protein spots which showed maternal or paternal inheritance of quantitative variations. These proteins were characterized by N‐terminal or internal amino acid sequencing, by determination of the amino acid composition, by glycoprotein staining and RNA expression analysis. The three proteins identified were: α‐enolase, cyclophilin and β‐group hemoglobins. The parental effects observed for α‐enolase and cyclophilin were found to be due to parent‐specific post‐translational modifications of these proteins. For the β‐group hemoglobins our results suggested parental effects on the transcriptional level.
Genotyping apolipoprotein B signal peptide insertion/deletion: A comparison of three methods
Authors: Glisic Sanja , Sunjevaric Ivana , Alavantic Dragan
Published: May 2005

Three different ways of detecting insertion/deletion (Ins/Del) genotypes of the apolipoprotein B gene encoding signal peptide were compared. Unequal quantities of the two alleles were observed in 50 Serbian subjects. The low abundance allele was clearly visible only when a modified method of silver staining for polyacrylamide gels was used. This method is more reliable for Ins/Del polymorphism genotyping, avoiding misclassifications by conventional ethidium bromide staining of both agarose and polyacrylamide gels.
From image processing to classification: II. Classification of electrophoretic patterns using self‐organizing feature maps and feed‐forward neural networks
Authors: Keşmir Can , Søndergaard Ib , Jensen Kirsten
Published: May 2005

In a recent study, isoelectric focusing patterns were classified with a neural network using the back‐propagation algorithm [1]. In order to further study the classification process and to generalize the presentation of electrophoretic patterns, Kohonen's self‐organizing feature maps [2] were applied in this study. Although these feature maps are very efficient in many pattern recognition tasks, our data proved to be too complex for classification with an unsupervised system. Therefore, a second supervised network on top of the feature map was necessary. As in [3], a feed‐forward network trained by the back‐propagation algorithm was used. The final system allows us to correctly classify 90% of all wheat varieties. Moreover, the system proved to be reliable, reasonable in training time and shows the same accuracy in different experimental setups.
Imaging electrophoretic gels with a scanning beam laser macroscope
Authors: Seto Eileen K. , Damaskinos Savvas , Dixon Arthur E. , Diehl‐Jones William L. , Mandato Craig A.
Published: May 2005

A scanning beam laser macroscope

Patents pending.

has been developed which scans an area of 7.5 × 7.5 cm in 5 s. This new imaging system is examined as a potential tool for scanning electrophoretic gels. A specially‐designed telecentric, ƒ*θ laser scan lens is used in the instrument to achieve a linear scan and a flat focal plane. The laser scan lens focuses the incoming beam from a laser to a 10 μm spot inside the gel. A raster scan is performed across the gel and the signal is detected with a photomultiplier, forming a 512 × 512 digital image stored as a computer file. Silver‐stained protein polyacrylamide gels have been imaged in transmission and double‐transmission, while DNA agarose gels (stained with ethidium bromide) have been imaged in fluorescence with better than 25 pg. sensitivity. The macroscope has the advantage that it is not tied to the electrophoresis system as are end‐of‐line scanners, and the scan is rapid, so that several gels can be scanned in a very short time.
Free flow‐isoelectric focusing of human cellular lysates as sample preparation for protein analysis
Authors: Burggraf Dorothe , Weber Gerhard , Lottspeich Friedrich
Published: May 2005

A whole cell lysate of human cells was separated into 80 fractions according to the pI of proteins using free flow isoelectric focusing with carrier ampholytes. The resolution of the process was highly reproducible, with an overlap of fractions of less than 30%. A protein of a faint silver stained spot in two‐dimensional gel electrophoresis (2‐DE) could be enriched, yielding a Coomassie blue stained spot which could be further characterized by proteinchemical methods. The enrichment of less abundant proteins from a complex crude cell extract was found to be a suitable tool for sample preparation and enrichment before applying proteins to 2‐DE and reversed‐phase high performance liquid chromatography.
Preparative reflux electrophoresis
Authors: Margolis Joel , Corthals Garry , Horvath Z. Stephen
Published: May 2005

As part of the method for large‐scale preparative electrophoresis across membranes of controlled porosity, we show that successive amounts of the leading component of a mixture migrating across a thin membrane can be collected by ‘reflux electrophoresis’. This consists of a series of cycles, in each of which the forward phase is stopped before the trailing components can emerge, the leading fraction is collected and the membrane cleared by reversing the current before commencing the next cycle. The reflux principle is demonstrated by separations based on size or on charge differences of protein molecules.
Separation of heat‐stable proteins from Thermus thermophilus HB8 by two‐dimensional electrophoresis
Authors: Kawaguchi Shin‐ichi , Kuramitsu Seiki
Published: May 2005

Thermostable proteins from Thermus thermophilus HB8, an extremely thermophilic bacterium, were separated by two‐dimensional gel electrophoresis. About 1200 spots were detected with silver staining on the gel between pH 3 and 10. According to the genome size of T. thermophilus, we consider that more than half of the proteins in the cell are visualized on a two‐dimensional gel. Using comigrated standard marker proteins, the molecular weight and isoelectric point of each protein spot were calculated. The average molecular weight and isoelectric point values were estimated to be 30 000 and 5.2, respectively. The average size and isoelectric point of detected protein from T. thermophilus were smaller and more acidic than those from Escherichia coli. After the protein spots had been electroblotted onto a polyvinylidene difluoride membrane and stained with Coomassie Brilliant Blue, the N‐terminal amino acid sequences were determined for about twenty protein spots. Few proteins had blocked N‐termini. Some spots were identified as proteins whose sequences had been reported previously from T. thermophilus. Others had amino acid sequences homologous with those of various proteins from other organisms. The amino acid sequence information of this report will be useful for obtaining stable proteins and for identifying open reading frames determined from the genome DNA sequence. Considering its small genome size and protein stability, T. thermophilus will be an excellent candidate for studying the molecular biology of an autotrophic living cell at the atomic level.