bluuebunny/crossref_metadata_2025 huggingface dataset:
Pouring wide‐range immobilized pH gradient gels with a window of extremely flattened slope
Authors: Altland Klaus , Altland Alexander
Published: May 2005

A gradient mixing device has been designed to pour polyacrylamide gels with a widerange immobilized pH gradient including a „window”︁ of extremely flattened slope. The device consists of an IBM‐compatible personal computer controlling 8 step‐motor‐driven burettes, with four producing a density gradient from glycerol and delivering acrylamides and catalysts for gel polymerization, two delivering Immobilines for a wide‐range pH gradient and the other two burettes responsible for the delivery of Immobilines for a partial pH range inside the wide range. The effect on a complex separation pattern of proteins with a wide range of pI is that resolution can be increased reproducibly to any reasonable extent at any location of the separation pattern.
Deoxyribonuclease zymography adapted to the precast PhastGel electrophoresis media
Authors: Strætkvern Knut O. , Raae Arnt J.
Published: May 2005

A set‐up for casting fluorescent indicator agarose gels on ultrathin polyacrylamide microelectrophoresis gel media (Pharmacia PhastGel media) is described. The zymogram system allows a rapid and sensitive detection of deoxyribonuclease in various gel media, following isoelectric focusing, native and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis.
Image analysis techniques for automatic evaluation of two‐dimensional electrophoresis
Authors: Häder Donat‐P. , Kauer Gerhard
Published: May 2005

Techniques for automatic analysis of two‐dimensional electrophoresis gels by computer‐aided image analysis are described. Original gels or photographic films are scanned using a laser scanner and the files are transferred to a microcomputer. The program package first performs a compression and preevaluation of the files. Spot identification and quantification is performed by the chain code algorithm after appropriate zooming and cutting. Labeling facilitates spot identification and quantification in numerical and graphical (pseudocolor) representation on peripheral devices for camera ready output. Interpolation between measured basepoints is performed by cubic spline algorithms which are automatically switched on and off, depending on the need by the program. High speed analysis and graphic representation is achieved using fast Assembler language routines rather than high level languages. One‐dimensional gels can be analyzed using the same software. Spot matching between parallel two‐dimensional gels has not yet been implemented.
A review of the CLIP system for the quantitative analysis of two‐dimensional electrophoresis gels
Authors: Potter David J.
Published: May 2005

This paper reviews the CLIP image processing system for the complete analysis of two‐dimensional electrophoresis images. The analysis problem for two‐dimensional gel images can be broken down into three issues: segmentation of individual gel images, alignment and comparison of pairs of gel images, and information storage and retrieval. This paper describes these problems and reviews how the CLIP system handles each of them. Segmentation is the location and isolation of each protein spot on an individual gel image and also the extraction of individual spot data such as position, area and volume. There are three basic stages: background field correction, noise filtering, spot detection and information extraction. Alignment and comparison of gel images involves matching protein spots between two gels. This can be quite difficult because there is not a simple relationship which can transform one gel image onto another. The database issues concern storing all the information which has been obtained from the above operations such that retrieval of this information can be readily performed. The advantage of the CLIP system over others is speed of processing. CLIP series computers use one processor for every pixel of the camera image such that image processing algorithms run in parallel. The main disadvantage is in the cost of these machines. With the declining trend in the cost of parallel processors, these machines will become more and more viable alternatives. This papers reviews the algorithms for the analysis of two‐dimensional gels. It is shown that CLIP is flexible enough to perform more than one type of algorithm for a particular operation.
Scale‐up of free flow electrophoresis: I. Purification of alcohol dehydrogenase from a crude yeast extract by zone electrophoresis
Authors: Hoffstetter‐Kuhn Sabrina , Wagner Horst
Published: May 2005

The potential and limitations in scaling‐up free‐flow electrophoresis, with emphasis on zone electrophoresis, are demonstrated. Purification of alcohol dehydrogenase (ADH) from a crude yeast extract was chosen as a model for an industrial approach to enzyme purification. In zone electrophoresis the separation quality strongly depends on the pH and conductivity of the background electrolyte, its residence time and flow rate, as well as the applied voltage. Optimization of these parameters resulted in a purification factor of 5.3 and a yield of 96% ADH, using a Tris/HCl buffer, pH 8.0, and a conductivity of 1 mS/cm, with a residence time of 10 min at 500 V. The loading capacity of the method for a laboratory‐sized free‐flow electrophoresis apparatus was limited to a sample throughput of about 0.4 g/h. By increasing the chamber dimensions it was possible to purify the enzyme by a purification factor of 4.7 and a yield of 93% ADH, at a throughput of about 1 g total protein/h. By simultaneously applying the sample at 3 input positions the throughput could be increased to 2.75 g/h with a purification factor of 4.7 and an overall yield of 90%.
Analysis of 6‐phosphogluconate dehydrogenase isozymes by polyacrylamide gel isoelectric focusing
Authors: Akiyama Katsunori
Published: May 2005

Three common phenotypes of 6‐phosphogluconate dehydrogenase (6‐PGD) were identified by enzyme visualization following isoelectric focusing in photochemically polymerized polyacrylamide gels whereas in chemically polymerized gels no activity could be detected. Gene frequencies calculated from 429 individuals were 0.915 and 0.085 for 6‐PGD*A and 6‐PGD*C, respectively, similar to the gene frequencies of Japanese samples previously reported.
Two‐dimensional electrophoresis of dog bronchoalveolar lavage fluid proteins
Authors: Lenz Anke‐Gabriele , Meyer Barbara , Weber Hans , Maier Konrad
Published: May 2005

Proteins of dog bronchoalveolar lavage fluid, obtained by washing the epithelial lining layer of lungs with phosphate‐buffered saline, were separated by two‐dimensional electrophoresis. Due to the low protein and high salt content of the bronchoalveolar lavage fluid, samples had to be concentrated and desalted. Following electrophoresis the protein spots were visualized by silver staining. Comparing the two‐dimensional protein patterns of bronchoalveolar lavage fluid with that from serum, several lungspecific proteins were detected. The most prominent protein, most probably a surfactant‐associated protein, showed isoforms with isoelectric points in the range of pH 4.2–4.8, and a molecular mass of 32 kDa on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis after reduction with dithiothreitol.
Internal sequence analysis of proteins separated on polyacrylamide gels at the submicrogram level: Improved methods, applications and gene cloning strategies
Authors: Tempst Paul , Link Andrew J. , Riviere Lise R. , Fleming Mark , Elicone Christopher
Published: May 2005

The fields of protein chemistry and molecular biology are currently merging for study of biologically relevant events and conditions. To obtain partial sequences of microamounts of protein, effcient integration of high resolution separation and sequencing technologies is required. We report here on improved methods that allow extensive internal sequencing of 10 to 20 picomoles protein recovered from one‐ or two‐dimensional gels. Each step of the standard protocol of Aebersold et al. (Proc. Natl. Acad. Sci. USA 1987, 84, 6970–6974) and the required instrumentation were examined and specifically adapted for use with submicrogram amounts of protein. Optimizations of in situ microdigests and liquid chromatography were needed for improved peptide recovery. Subsequent automated sequencing required subpicomole analysis. New methods for S‐alkylation of gel‐separated proteins and accurate identification of tryptophan‐containing peptides were introduced to insure over all higher efficiencies. The acquired internal sequences facilitated cloning of the genes and several strategies are discussed. Applying our method, several proteins of unknown structure were sequenced and successfully identified or cloned. Internal sequences of submicrogram protein amounts, recovered from a single two‐dimensional gel of Escherichia coli total protein (120 μg), allowed unambiguous identification of the spots but pre‐gel enrichment will be required for analysis of most (90–95 %) other spots. Integration of comprehensive two‐dimensional gel protein databases with methods and strategies outlined here could potentially be an abundant source of DNA probes and markers useful for guidance of the human genome sequencing project and for analysis of the emerging vast amounts of data.
Electroblotting of polypeptides onto glass fiber filters for direct sequence analysis after sodium dodecyl sulfate‐polyacrylamide gel electrophoresis
Authors: Bergman Tomas , Jörnvall Hans
Published: May 2005

The technique of electroblotting polypeptides onto Polybrene‐treated glass fiber filter discs after protein detection with potassium chloride is evaluated further with different proteins in separate applications. The number of proteins analyzed with this method is now more than double that previously reported. Reproducible results in good yield are obtained. Average overall yield – including the electrophoretic step before blotting – is 26 %, with maximal recoveries through all steps up to 60 %. High sensitivity radiosequence analysis is also applicable. Recent modification of the previously described procedure include use of Whatman glass fiber filters, removal of air in the Polybrene‐impregnated filters by buffer penetration under reduced pressure, and use of widely different times for electrotransfer. Special advantages with this method are low extent of protein α‐amino group destruction, direct use of the entire filter in the sequencer, and insensitivity to variations in electroblotting time. Gas‐phase hydrolysis in situ of blotted proteins followed by amino acid analysis is known to give a low yield of polar amino acids, and often artifacts, but can still give an estimate of the amount of polypeptide immobilized on the filter. A wash with n‐butyl chloride is now shown to reduce the Polybrene‐associated artifacts, and an addition of sodium chloride to increase the recovery of polar amino acids. These two steps therefore appear interesting in schemes for compositional analyses of electroblotted proteins.
Identification of rat liver glutathione S‐transferase Yb subunits by partial N‐terminal sequencing after electroblotting of proteins onto a polyvinylidene difluoride membrane from an analytical isoelectric focusing gel
Authors: Chang Li‐Hsueh , Hsieh Jyh‐Chang , Chen Woan‐Ling , Tam Ming F.
Published: May 2005

Rat liver glutathione S‐transferases were partially purified using S‐hexyl glutathione affinity chromatography, followed by native isoelectric focusing employing a pH 7–11 or pH 3–10 gradient. Proteins were excised and eluted from the gel for determination of subunit composition using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. In separate experiments, isoelectric focusing gels were equilibrated with a sodium dodecyl sulfate‐containing buffer at high pH, and proteins on the gel were electroblotted onto a polyvinylidene difluoride membrane, utilizing graphite plates as electrodes. The membrane‐bound proteins were visualized by Coomassie Brilliant Blue staining. The protein bands were then excised from the membrane and inserted into a gas phase sequenator for direct sequencing. N‐Terminal sequences thus determined were compared with published cDNA sequences. The isoelectric points (pIs) and positions on the isoelectric focusing gel of Yb1 Yb1, Yb1 Yb2 and Yb2 Yb2 subunits were determined. We have also located on the pH 3–10 focusing gel an N‐terminal blocked glutathione S‐transferase which has a molecular weight similar to Yb subunits.
Capillary isotachophoresis with UV and tandem mass spectrometric detection for peptides and proteins
Authors: Smith Richard D. , Fields Steven M. , Loo Joseph A. , Barinaga Charles J. , Udseth Harold R. , Edmonts Charles G.
Published: May 2005

The application of capillary isotachophoresis (CITP) and combined CITP‐mass spectrometry (MS) for peptides and proteins is demonstrated. Separation of simple peptide mixtures, as well as enzymatic digets of proteins, is also reported using CITP with UV detection. The potential utility of CITP for proteins is demonstrated. Initial studies of combined CITP‐MS of enzymatic digests is also demonstrated, showing the potential for rapid sequence determination.
Mathematical description of electrophoretic separation of weak acids by means of an H+ pulse
Authors: Gebauer Petr , Deml Mirko , Pospichal Jan , Boček Petr
Published: May 2005

The paper gives a detailed theoretical description of the recently published principle of separation by a dynamic pulse in zone electrophoresis. The theory is illustrated by using a system in which weak acids are separated by migrating against an H+ pulse. The mathematical description pertains to (i) the evolution and migration of the concentration profile of the H+ pulse, and (ii) the migration of a sample substance (here a weak acid) against and through the pulse. As a result the migration (detection) times of the separated sample components are obtained. Based on the theory, a graphical treatment was developed which allows fast optimization of the separation conditions by observing (on the PC monitor) how the trajectories of the substances (drawn in a time vs. distance coordinate system) change with a change in the parameters of the pulse. Some of the results illustrate the possible advantages of using the dynamic pulse method in analytical practice.
Recent advances in capillary electrophoresis of proteins, peptides and amino acids
Authors: Novotny Milos V. , Cobb Kelly A. , Liu Jinping
Published: May 2005

The current status of high‐performance capillary electrophoresis as an analytical separation method for proteins, peptides and amino acids is assessed. Recent advances in suppressing the effects of electroosmotic flow and irreversible adsorption of proteins at the capillary wall are reviewed, together with procedures for optimal separations of peptides and amino acids. The detection aspects emphasize the role of laser‐induced fluorescence and capillary electrophoresis/mass spectrometry in high‐sensitivity measurements.
Capillary zone electrophoresis of rare earth metals with indirect UV absorbance detection
Authors: Foret František , Fanali Salvatore , Nardi Annalisa , Boček Petr
Published: May 2005

The separation of 14 lanthanides by capillary zone electrophoresis was studied in the background electrolyte containing hydroxyisobutyric acid as complexing counterion and creatinine as a UV absorbing coion for indirect detection of lanthanide zones. A complete separation was achieved in less than 5 min and the applicability of the method for the analysis of real samples was demonstrated.
Analysis of the conformational transitions of proteins by temperature‐gradient gel electrophoresis
Authors: Birmes Andreas , Sättler Andrea , Maurer Karl‐Heinz , Riesner Detlev
Published: May 2005

Temperature‐gradient gel electrophoresis (TGGE) is a technique for studying the structural transitions of nucleic acids and proteins. A temperature gradient is formed in a horizontal slab gel perpendicular to the direction of the electric field. Whereas the principle of the TGGE method has previously been applied to proteins, we describe in this report the systematic optimization of TGGE as a routine technique for the quantitative analysis of conformational transitions in proteins. Using α‐amylase as an example we show the kinds of results which may be obtained from such measurements. Buffers suitable for use in gel electrophoresis were analyzed with respect to the dependence of their pH value upon temperature. The correct pH range for TGGE of a given protein is determined by electrophoretic titration curves. The protein bands are detected by silver and/or activity staining. The thermal denaturation of α‐amylase from Aspergillus oryzae showed a discontinuous transition into the denatured conformation, which exhibited much slower electrophoretic mobility. The discontinuity is due to an irreversible denaturation process under the gel conditions. The transition temperature was measured as a function of several parameters, e. g., the concentration of Ca++, dithiotreithol, urea and the pH value. The structural transition of α‐amylase is accompanied by a loss of enzymatic activity as determined by activity staining or by an activity assay carried out in solution. The structural transitions of two other α‐amylases from Bacillus subtilis and Bacillus licheniformis were also studied. The results show that the TGGE method is simple to perform and allows the analysis of conformational transitions of proteins in a wide variety of conditions. It is also possible to analyze the conformational stability of proteins in unpurified extracts if activity‐ or immuno‐tests are used for detection.
Electrophoretic and immunological evidence of unique proteins in leaves of citrus trees: Application to citrus blight detection
Authors: Bausher Michael G.
Published: May 2005

From the leaf tissue of healthy and blighted citrus trees 10–30 kDa soluble fractions were compared to find biochemical markers of tissue in the disease state. Using a non‐denaturing extrction technique coupled with ultrafiltration, a resulting 10–30 kDa healthy and citrus blight fraction was sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Two distinct and adjacent bands for blight at an Mr of approximately 12 500 were separated. These bands were visible with Coomassie Brilliant Blue and silver stain but were negative to glycoprotein stains. An antiserum prepared against proteins isolated by preparative electrophoresis reacted only with the blight fractions and was distinctly different from healthy fractions when Western blotted. Only the gel region (Mr 12 500–13 000) of citrus blight sources was positive to the antiserum when compared with disease and nondisease stress sources. Results indicate that identification of specific proteins may be a way to diagnose the onset of citrus blight prior to visible tree symptoms.
Orosomucoid typing by isoelectric focusing: Genetic variation of orosomucoid in Asian macaques (genus Macaca)
Authors: Yuasa Isao , Umetsu Kazuo , Shotake Takayoshi , Ishida Takafumi , Takenaka Osamu , Terao Keiji , Kawamoto Yoshi
Published: May 2005

Genetic variation of orosomucoid (ORM) in the genus Macaca was investigated Plasma samples were subjected to isoelectric focusing in a pH range of 4–6.5, followed by immunoprinting with anti‐human ORM antibodies. A total of 25 allele were identified in 231 Asian macaques belonging to 13 species from 23 population and 22 members belonging to a family of M. fascicularis. Family data presented evidence for a codominant mode of inheritance with multi‐alleles at a single auto somal locus. A population study revealed enormous intra‐ and interspecies variations. The heterozygosity values varied from 0.855 in M. fascicularis (Malaysia) to 0.000 in M. radiata (India), M. silenus (India) and M. arctoides (Malaysia).
Discontinuous polyacrylamide gel ‐ agarose gel immunoelectrophoresis for analysis of plasma lipoproteins
Authors: Dumon Marie‐France , Camare Rita , Nassar Brahim‐Bertrand , Dang Quoc‐Quan , Clerc Michel
Published: May 2005

A modification of crossed immunoelectrophoresis for the analysis of plasma lipoproteins is described and is called polyacrylamide gel‐crossed immunoelectrophoresis. The incorporation of albumin in the first‐dimensional gel facilitates the transfer of the larger lipoproteins containing apolipoprotein B from the first‐dimensional gel to the second dimension. Furthermore, under this condition the quantitation of total apolipoprotein B by polyacrylamide gel‐crossed immunoelectrophoresis is in good agreement with the results obtained by rocket immunoelectrophoresis or nephelometry. The correlation between polyacrylamide gel‐crossed immunoelectrophoresis and rocket immunoelectrophoresis is good for total apolipoprotein B (p > 0.001) and apolipoprotein A‐I (p > 0.001). Polyacrylamide gel‐crossed immunoelectrophoresis also offers interesting aspects to study the plasma lipoprotein classes and subclasses in different cases: normal plasma, current and complex dyslipoproteinemias in the presence or absence of lipoprotein small a. In a case of dyslipoproteinemia of Fredrickson′s Type V polyacrylamide gel‐crossed immunoelectrophoresis demonstrates the presence of a small‐sized Lp (a) major peak in the low density lipoprotein (LDL) zone and of a large‐sized Lp (a) minor peak in the very low density lipoprotein (VLDL) zone.
Intensification of peroxidase‐diaminobenzidine staining using gold‐sulfide‐silver: A rapid and highly sensitive method for visualization in immunoblotting
Authors: Iida Reiko , Yasuda Toshihiro , Nadano Daita , Kishi Koichiro
Published: May 2005

A highly sensitive and rapid visualization method for protein detection by immunoblotting is described. Proteins blotted onto a Durapore membrane were visualized by the following procedure: after conventional peroxidase‐based staining with 3,3′‐diaminobenzidine (DAB), the produced DAB precipitates were intensified by treating with (i) gold trichloride (acid), (ii) sodium sulfide, and (iii) a developer containing silver nitrate. This postintensification method was employed for the detection of the genetic polymorphism of human proteins, such as deoxyribonuclease I in urine, and group specific component, transferrin and α1‐antitrypsin in serum after polyacrylamide gel‐isoelectric focusing, followed by immunoblotting. This postintensification technique was found to be simple, giving up to 16‐ to 64‐fold amplification of the conventional peroxidase‐DAB staining.
Recovery of the contact site A glycoprotein of Dictyostelium discoideum from sodium dodecyl sulfatepolyacrylamide gel and characteristics of monoclonal antibodies against the recovered protein
Authors: Ochiai Hiroshi , Jin Kimiaki , Kakihara Noriaki , Saito Tamao , Fukuzawa Masashi
Published: May 2005

Monoclonal antibodies were produced against a cell‐cell adhesion (contact site A) glycoprotein of Dictyostelium discoideum, isolated by preparative gel electrophoresis. The glycoprotein was recovered by electroelution from a polyacrylamide gel strip and used for the production of monoclonal antibodies. Four of the five antibodies obtained bound specifically to the protein moiety of the contact site A glycoprotein. The specificities of the antibodies were in striking contrast to those of antibodies raised against the contact site A glycoprotein purified by Triton X‐114 phase separation and DEAE chromatography. The majority of the latter antibodies recognized the carbohydrate moiety of the contact site A glycoprotein and cross‐reacted heavily with other membrane glycoproteins.
A general‐purpose pulsed field controller
Authors: Abbal Philippe , Picou Christian
Published: May 2005

A pulsed field controller compatible with various types of electrophoresis tanks and power supplies is described. The system is based on a high‐precision timing circuit, with an output isolated from the main high voltage supply, and four high voltage transistors. The switching time is programmed manually between 5–160 s. With a transverse alternating field electrophoresis system, intact yeast chromosomes could be completely separated after 24 h.
Purification of S1 nuclease from Aspergillus oryzae by recycling isoelectric focusing
Authors: Ostrem James A. , Van Oosbree Thomas R. , Marquez Rodolfo , Barstow Leon
Published: May 2005

We describe the purification of a single‐strand nuclease from Aspergillus oryzae using the first commercial prototype of an instrument (RF3TM) designed by Milan Bier et al. for preparative‐scale insoelectric focusing. Protein separation takes place entirely in solution, with shear‐stabilized laminar flow counteracting convective disturbances generated by the electric field. Conditions for isoelectric focusing were determined by focusing fractions with nuclease activity, following chromatography on DEAE‐Sepharose, in analytical gels containing carrier ampholytes. The separation was then scaled up to process larger quantities of protein in the RF3. When partially‐purified protein (250 mg, 6700 U/mg) was focused in pH 3‐4 carrier ampholytes, 67% of the activity was recovered in pooled peak fractions with a specific activity of 54 000 U/mg protein. Overall, 82% of the activity loaded on the RF3 was recovered. Eliminating two steps prior to isoelectric focusing, and increasing the protein load from 250 mg to 1.2 g, produced an enzyme with a nearly four‐fold increase in specific activity (from 4000 U/mg protein to 15 000 U/mg protein) but with unacceptable color. Our results indicate that a high quality enzyme can be prepared in quantity when heat denaturation and ammonium sulfate precipitation are included prior to isoelectric focusing.
Biomedical and clinical applications of automated single cell electrophoresis
Authors: Schütt Wolfgang , Thomaneck Uwe , Knippel Eberhard , Rychly Joachim , Klinkmann Horst
Published: May 2005

The automated single cell electrophoresis microscope Parmoquant has been used for the discrimination of lymphocytes and the study of the interaction of substances with cells and synthetic particles. Electrophoretic histograms allowed the determination of changes in the proportion of lymphocyte populations after kidney transplantation, during dialysis treatment, open heart surgery and during pregnancy. Discrimination of leukemic cells on the basis of electrophoresis was used as an additional parameter in diagnosis. In a mouse tumor model, histogram determination enabled the in vivo effect of tumor necrosis factor on immune cells to be evaluted. Cell electrophoresis was shown to be suitable to detect the influence of antibodies, lectins and bacteria on the cell surface. Protein adsorption was studied on synthetic particles using cell electrophoresis. This method was applied to investigate the phenomena of blood interaction with biomaterials used in artifical organs and to determine differences in the protein composition of serum or other body fluids connected with diseases.
Separation of rat pituitary secretory granules by continuous flow electrophoresis
Authors: Hayes Daniel , Exton Carrie , Salada Thomas , Shellenberger Kathy , Waddle Jenny , Hymer W. C.
Published: May 2005

The separation of growth hormone‐containing cytoplasmic secretory granules from the rat pituitary gland by continuous flow electrophoresis is described. The results are consistent with the hypothesis that granule subpopulations can be separated due to differences in surface charge; these, in turn, may be related to the oligomeric state of the hormone.
Preparative separation of pyrogens from proteins by isoelectric focusing using a multicompartment electrolyser with Immobiline membranes
Authors: Lucas J. , Faupel M. , Goecking C.
Published: May 2005

Due to the nature of lipopolysaccharide endotoxin structures of bacterial pyrogens, their removal from solutions containing therapeutic proteins is often a problem in the pharmaceutical industry. In this report we describe the application of electromotive force to dislodge lipopolysaccharide endotoxins from proteins. This was performed by employing a multicompartment electrolyzer fitted with Immobiline membranes of specified pIs. A thousand‐fold reduction of endotoxin could be achieved in the model test system described. This contribution describes the use of a new recycling isoelectric focusing approach without the use of carrier ampholytes.
Denaturing gradient gel electrophoretic analysis of minisatellite alleles
Authors: Uitterlinden André G. , Vijg Jan
Published: May 2005

By two‐dimensional DNA fingerprinting, an electrophoretic method which combines separation according to size with separation in a denaturing gradient, virtually all minisatellite sequences detected with a minisatellite core probe can be resolved (Uitterlinden et al., Proc. Natl. Acad. Sci. USA. 1989, 86, 2742–2746). To investigate the electrophoretic behavior in denaturing gradient gels of allelic restriction fragments containing minisatellite sequences, we analyzed alleles of the two highly polymorphic minisatellite loci D7S22 and D2S44. The results obtained indicate that for these loci, depending on the restriction enzyme used to digest genomic DNA, alleles of different sizes migrate to regions of similar denaturant concentration, i.e. to isothermal positions in the denaturing gradient. Denaturing gradient gel electrophoresis also allows for the discrimination of restriction fragments which are the result of the presence of internal recognition sites in the minisatellite and, therefore, to distinguish between VNTR and restriction site polymorphisms.
Detection of β‐glucanase activity on various β‐1,3 and β‐ 1,4‐glucans after native and denaturing polyacrylamide gel electrophoresis
Authors: Côté François , El Ouakfaoui Souad , Asselin Alain
Published: May 2005

β‐Glucanases were detected after polyacrylamide gel electrophoresis under native and denaturing conditions using various β‐1, 3‐ and β‐1, 4‐glucans, including mixed glucans (laminarin, pachyman, carboxymethyl cellulose, lichenan and barley β‐glucan). After electrophoresis and incubation of gels, substrates incorporated into polyacrylamide gels were stained with specific fluorochromes, Sirofluor for β‐1, 3 linkages and Calcofluor White M2R for β‐1, 4 linkages. Under UV illumination, lysis zones appeared as dark bands against a fluorescent background. Enzymes of bacterial, fungal and plant sources could be revealed sequentially in gles containing mixed β‐(1.3) (1,4)‐glucans by staining first with sirofluor followed by staining with Calcofluor White M2R. Active profiles were more diverse when substrates were stained with sirofluor. The use of purified sirofluor at pH 11.5 compared with Aniline Blue at pH 8.6 allowed better detection of β‐1, 3‐glucanase activities. In gels containing laminarin stained with sirofluor, bands exhibiting a more intense fluorescence than the background fluorescence were observed in addition to dark nonfluorescent bands. It is postulated that these two types of β‐1, 3‐glucanase activities differ by their enzymatic action (partial versus extensive hydrolysis). Analysis of fungal extracts using denaturing gels embedded with various β‐glucans displayed lysis bands migrating between 32 and 35 kDa.
Cationic detergent in agarose for improved electrophoresis of cationic proteins
Authors: Reimert Claus M. , Bøg‐Hansen Thorkild C.
Published: May 2005

Inclusion of the cationic detergent N‐cetyl‐N,N,N‐trimethylammonium bromide (CTAB) in normal agarose gels increases the electrophoretic separation and banding pattern of cationic proteins. This is shown for an extract of cationic granular proteins from polymorphonuclear granulocytes.
Oligonucleotide fingerprinting with (CAC)5: Nonradioactive in‐gel hybridization and isolation of individual hypervariable loci
Authors: Zischler Hans , Hinkkanen Ari , Studer Roland
Published: May 2005

The first topic to be treated in this paper is the nonradioactive DNA fingerprinting by means of in‐gel hybridization with digoxigenated (CAC)5. Besides the fact that timeconsuming Southern blotting can be avoided, the dried agarose is an excellent matrix to produce background‐free nonradioactive DNA fingerprints. There is no tendency of either the oligonucleotide probe or the antibody towards unspecific binding to the dried agarose. Prehybridization and blocking steps are therefore superfluous. Furthermore, we will discuss what effect the degree of crosslinking of the antibodyenzyme conjugates has. The second topic concerns the isolation and characterization of locus‐specific probes from a human (CAC)5 fingerprint. The isolation and characterization of one variable probe, by screening complete genomic libraries, is described and discussed. This probe is compared to a hypervariable single‐copy probe, isolated from a size‐enriched genomic library. The sequence of the repeat flanking locus‐specific probe is presented and a semi‐specific, adaptor‐mediated polymerase chain reaction was designed to amplify (CAC)n/(GTG)n flanking sequences.
Databases, quality control and interpretation of DNA profiling in the Home Office Forensic Science Service
Authors: Gill Peter , Evett Ian W. , Woodroffe Susan , Lygo Joan E. , Millican Emma , Webster Mark
Published: May 2005

The history of DNA profiling in the Home Office Forensic Science Service began with the introduction of multilocus probes into casework in 1986. The use of singlelocus probes was introduced in 1990, supported by databases of three ethnic groups; interpretation is backed up using a Bayesian approach. Databases were compiled using an image analysis computing system. Quality control systems are described, detailing requirements before a sample can be included in the database.
A discussion of the robustness of methods for assessing the evidential value of DNA single locus profiles in crime investigations
Authors: Evett Ian W. , Gill Peter
Published: May 2005

We discuss three issues relating to the evaluation of DNA single locus probe comparisons in forensic casework: band shift, database size, and population stratification. Illustrations are from experiments carried out in the Forensic Science Service as part of our preparations to implement the technique.
Secretion of lipid‐poor nascent human apolopoprotein apoAI, apoCIII, and apoE by cell clones expressing the corresponding genes
Authors: Hussain M. Mahmood , Roghani Ali , Cladaras Christos , Zanni Eleni E. , Zannis Vassilis I.
Published: May 2005

The human apolipoprotein apoAI, apoCIII, and apoE genes were placed under the control of the mouse metallothionein 1 promoter in a bovine papilloma virus vector that also contained the human metallothionein IA gene. Following transfection of mouse C127 cells with the expression vector, cell clones resistant to Cd2+ were selected and found to express in high abundance specific apolipoprotein genes. Individual cell clones expressing apoAI, apoCIII, or apoE genes were used further to study the isoprotein composition and the flotation properties of the corresponding nascent apolipoproteins. It was found that the lipoproteins secreted by cell clones ex‐pressing the apoAL, apoCIII, and apoE genes consisted of the proapoAI disialylated form of apoCIII (apoCIIIs2) and mainly sialylated forms of apoE. Separation of the secreted apolipoproteins by density gradient ultracentrifugation resulted in limited flotation of nascent apoAI, apoE and apoCIII in the high density lipoprotein (HDL) fraction. Similar analysis in the presence of human serum increased the flotation of apoAI, apoE, and apoCIII to 6.5‐, 4.5‐, and 5.5‐fold, respectively, and resulted in their redistribution to various lipoprotein fractions. HDL increased the flotation of apoAI to 12‐fold and very low density lipoprotein (VLDL) increased the flotation of apoCIII and apoE to 6.5‐ and 5.5‐fold, respectively. These findings suggest that in the cell system used, the majority of nascent apoAI, apoCIII and apoE is secreted in the lipid‐poor form, which then associates extracellularly with preexisting lipoproteins.
A reliable two‐dimensional gel electrophoresis procedure for separating neural proteins
Authors: Semple‐Rowland Susan L. , Adamus Grazyna , Cohen Robert J. , Ulshafer Robert J.
Published: May 2005

A facile two‐dimensional gel electrophoresis procedure has been developed for the analysis of neural tissue proteins which eliminates the serious problems associated with protein insolubility at the point of sample application onto polymerized first dimension isoelectric focusing gels. This was accomplished by combining the methods of two previously published procedures. Our proecedure provides an alternative method to the complex gel systems often employed for less soluble proteins, and yields very reproducible, high resolution separations. This procedure, which is in routing use in our laboratories for the analysis of total proteins extracted from retina and brain, produces protein patterns that are easily compared using both visual and computer‐assisted image analysis techniques. Presented here are the results of a set of experiments designed to identify proteins unique to retina. This procedure should be useful to investigators studying protein changes resulting from genetic mutation, development, drug treatment or disease, in neural tissue as well as in virtually all other tissues.
Barley cultivar discrimination: I. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and glycoprotein blotting
Authors: Weiss Walter , Postel Wilhelm , Görg Angelika
Published: May 2005

Two different methods of detecting electroblotted glycoproteins after sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of Tris‐buffer soluble barley seed proteins were examined for their applicability for barley cultivar discrimination. These are the highly specific, lectin‐based concanavalin A/peroxidase method and the more general periodate/danyslhydrazine method. The results of the periodate/dansylhydrazine method enabled us to divide the 20 examined cultivars into three groups, whereas the more sensitive concanavalin A/peroxidase method revealed six different glycoprotein patterns. In comparison, sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and silver staining of the alcoholsoluble barley seed proteins (hordeins) gave nine different banding patterns. A combination of hordein electrophoresis together with glycoprotein staining by the concanavalin A/peroxidase method made it possible to classify the cultivars into twelve groups, the largest of which contained four cultivars. The qualitative expression of the glycoprotein patterns seemed to be independent of growth conditions, whereas the band intensities obviously were not. As a whole, glycoprotein blotting is a valuable supplement to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of hordeins in barley cultivar discrimination.
Gel electrophoresis of polystyrene particles in glutaraldehyde crosslinked polyvinyl alcohol
Authors: Pospichal Jan , Tietz Dietmar , Ittyerah T. Roy , Halpern David , Chrambach Andreas
Published: May 2005

Polystyrene sulfate and carboxylate particles (19–189 m radius) were subjected to electrophoresis in glutaraldehyde crosslinked polyvinyl alcohol of molecular weight 25.000 and 650.000 Da at various concentrations. The degree of crosslinking is severely limited by the mechanical properties of the gels that deteriorate beyond a glutaraldehyde concentration which decreases with increasing polyvinyl alcohol chain length. The effective fiber radius of the short‐chain and long‐chain polymer fiber was 45 ± 25 and 131 ± 47 nm, respectively. Thus, these media do not significantly exceed the apparent fiber thicknes of agarose, are more difficult to prepare – but are well‐defined synthetic product rather than natural ones, and have the advantage of carrying no net charge and can therefore be expected to exhibit no electroendosmosis.
Observation of orientation and relaxation of protein‐sodium dodecyl sulfate complexes during pulsed intermittent field polyacrylamide gel electrophoresis
Authors: Brassard Éric , Turmel Chantal , Noolandi Jaan
Published: May 2005

Polyacrylamide gel electrophoresis (PAGE) of proteins denatured with SDS (sodium dodecyl sulfate) has been used successfully to separate proteins according to their molecular mass. In spite of the extensive use of this technique, the motion of the protein‐SDS complex in a polyacrylamide gel is still not understood. Here we report on the observation of the orientation (in the field direction) and relaxation of protein‐SDS complexes during pulsed intermittent field PAGE experiments. The results give an indication of the stiffness of the molecules and may be useful for the development of a technique to improve the separation of large proteins using pulsed electric fields.
A miniaturized electrode configuration for isoelectric focusing
Authors: Megow Dirk , Jacobasch Gisela
Published: May 2005

An electrode configuration is described which allows fast isoelectric focusing (IEF) with conventional IEF systems. The equipment, which can be fixed on the cooling plate of a conventional IEF system, consists of a base plate on which flappable electrode holders are fastened. The handling is simple and needs only little time. Graphite rods are used as electrodes, thus avoiding the use of buffer strips. Samples are applied with special applicator strips – permitting the analysis of up to 19 samples on a 50 × 40 mm polyacrylamide gel and up to 44 samples on a 100 × 70 mm gel. Only 30 min are needed for one IEF run.
A novel method for specific visualisation of serum albumin in polyacrylamide gels by iodine staining
Authors: Lee Kuo‐Kau , Ellis Anthony E.
Published: May 2005

Bovine serum, bovine serum albumins (delipidated or globulin free or Fraction V), rabbit serum, rabbit serum albumin, Atlantic salmon serum, purified Atlantic salmon serum albumin, human plasma α2‐macroglobulin, hemocyanin, trypsin inhibitor, bovine transferrin and bovine lactoferrin were examined by a novel method for specific visualisation of albumins. In native polyacrylamide gel electrophoresis the albumins were visualised by iodine staining as a transparent spot against a brown background whilst the other proteins could not be visualised. It is suggested that the brown background was due to penetration of the gel by iodine while the chemical binding of iodine by albumin produced a decolourisation reaction. This novel method provides a fast and simple approach to identifying serum albumin in polyacrylamide gels.
A rapid (< 10 minute) electrophoresis method for identification of wheat varieties
Authors: Wrigley Colin W. , Gore Peter J. , Manusu H. Perry
Published: May 2005

Conventional procedures for electrophoretic identification of gain samples according to variety are too slow to permit checking at the time of delivery. The method described permits electrophoretic identification within an hour. It involves extraction of gliadin proteins from crushed grain with 6% urea solution or ethylene glycol, cathodic electrophoresis for 9 min at 300 V in a Micrograd gel (MG 315 from Gradipore Ltd, Sydney, Australia) using sodium lactate buffer (pH 3.1), and staining in Gradipore (at about 50°C). Distinction between a set of Australian varieties was similar to that obtainable with the Australian Standard Procedure.
Counterflow affinity isotachophoresis on cellulose acetate membranes
Authors: Schranz Dorota , Morkowski Stanislaw , Karamova Eleonora R. , Abelev Garri I.
Published: May 2005

Counterflow isotachophoresis on cellulose acetate membranes of human alphafetoprntein (AFP) was performed with concanavalin A, lentil lectin, and castor bean lectin driven by electroendosmotic counterflow. This counterflow caused a uniform stream of lectin to migrate towards the cathode against AFP with carrier ampholytes in steady‐state position. Retardation of microheterogeneity forms bound to lectins was observed, giving results comparable to standard crossed affinity immunoelectrophoresis. Smaller amounts of lectins and more diluted samples of AFP could be used in the described method.
Analysis of the interaction between human plasma fibronectin and gelatin by affinity electrophoresis
Authors: Kashiwagi Shiro , Nakamura Kazuyuki , Takeo Kazusuke , Takasago Teiichi , Uchimichi Akihiko , Ito Haruhide
Published: May 2005

The interaction between human plasma fibronectin and gelatin was analyzed by affinity electrophoresis, in which the fibronectin was subjected to electrophoresis in a 4% polyacrylamide gel in the presence and absence of gelatin, as an affinity ligand, and the fibronectin band was stained by an immunoblotting method. The apparent dissociation constants (Kd) of fibronectin for gelatin were calculated from affinity plots based on the original affinity equation at different pHs, urea concentrations, and temperatures. The fibronectin exhibited much lower affinity in the presence of urea. The Kds at 37°C were 1.49 × 10−7 M, 2.50 × 10−6 M, and 3.58 × 10−6 M with 2 M, 3 M, and 4 M urea, respectively. The van't Hoff plots of Kd values against absolute temperature (T) showed that the value of log Kd decreased in proportion to the increase in the value of 1/T within the range of 15–50°C. The standard enthalpy, the standard free energy change at 37°C, and the entropy change at 37°C for association were calculated to be −124.7 kJ/mol, −33.23 kJ/mol, and −295.1 J/mol/deg, respectively. These results suggest that a hydrophilic interaction, such as hydrogen bond or van der Waals interaction, plays an important role in the binding of plasma fibronectin to gelatin.
Demonstration of murine pancreas elastase and its interstrain variation by isoelectric focusing
Authors: von Deimling Otto H. , Gaa Andreas , Hameister Horst
Published: May 2005

Isoelectric focusing between pH 9 and 11 was used for separation of murine pancreas proteases. One of these proteases is characterized by its preference for N‐acetyl‐L‐alanine‐α‐naphthylester as substrate and by its genetic linkage to bt, a coat color marker of chromosome 15. This protease was identified as elastase, and is probably elastase‐1 (ELA‐1). Because of the simple procedure and the excellent reproducibility of the focusing pattern, ELA‐1 is recommended as a useful marker for mouse chromosome 15.
Analysis of cerebrospinal fluid from patients with psychiatric and neurological disorders by two‐dimensional electrophoresis: Identification of disease‐associated polypeptides as fibrin fragments
Authors: Wildenauer Dieter B. , Körschenhausen Dirk , Hoechtlen Wilma , Ackenheil Manfred , Kehl Maria , Lottspeich Friedrich
Published: May 2005

Two‐dimensional electrophoresis (2‐DE) of cerebrospinal fluid (CSF) samples–from 347 patients with various psychiatric and neurological disorders–and subsequent silver staining revealed two additional polypeptides (Mr 40000) in 49% of 111 schizophrenics, 46 % of 43 schizoaffective patients, 36 % of 41 patients with affective disorders, 43 % of 28 patients with multiple sclerosis, but not in 25 patients without neurological symptomatology, nor in 9 patients with Lues, and in only 2 of 25 patients with AIDS. The two polypeptides, as detected by 2‐DE, eluted after size exclusion chromatography in fractions containing proteins with Mr > 200000. After 2‐DE of CSF samples, enriched by gel chromatography, the polypeptides were immobilized by blotting onto glass‐fiber membranes and subjected to N‐terminal sequencing. Polypeptide A was identified as β‐chain remnant (β2), derived from plasmin cleavage of fibrin(ogen). After size exclusion chromatography, 2‐DE, and Western blotting, polypeptide A and B, as well as several other spots, reacted with fibrinogen antibodies, suggesting that the polypeptides are subunits of a fibrin degradation complex.