bluuebunny/crossref_metadata_2025_split huggingface dataset:
A review of the CLIP system for the quantitative analysis of two‐dimensional electrophoresis gels
Authors: Potter David J. 
Published: May 2005

AbstractThis 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

AbstractThe 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

AbstractThree 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

AbstractProteins 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

AbstractThe 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

AbstractThe 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

AbstractRat 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

AbstractThe 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

AbstractThe 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

AbstractThe 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

AbstractThe 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

AbstractTemperature‐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

AbstractFrom 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

AbstractGenetic 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

AbstractA 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

AbstractA 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

AbstractMonoclonal 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

AbstractA 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

AbstractWe 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.