bluuebunny/crossref_metadata_2025_split huggingface dataset:
Capillary enzymophoresis of nucleic acid fragments using coupled capillary electrophoresis and capillary enzyme microreactors having surface‐immobilized RNA‐modifying enzymes
Authors: Mechref Yehia , El Rassi Ziad 
Published: May 2005

AbstractThis report describes the coupling of capillary enzyme reactors to capillary electrophoresis, which is termed capillary enzymophoresis. In the present study, the capillary enzyme reactors were prepared by immobilizing RNA‐modifying enzymes, e.g., RNase T1 and RNase U2, on the inner walls of 50 μm fused‐silica capillaries. These microreactors served to selectively modify the solutes (or substrates) before entering the separation capillary. Capillary enzymophoresis using single or mixed enzyme reactors proved useful in identifying minute amounts of dinucleotides as well as the fingerprinting of tRNAs. The immobilized RNase T1 and RNase U2 displayed their usual enzymic activities toward RNA fragments and in addition exhibited different activity‐pH dependency than the soluble enzymes. This was attributed to microenvironmental effects arising from the charged nature of the capillary walls in the close proximity of the immobilized enzymes. The enzyme reactors were reusable for several RNA samples and showed chemical and thermal stabilityPresented as a part of a lecture (#313) at Pittsburgh Conference 94 (Pittcon '94), Chicago, Illinois, February 27‐March 4, 1994.
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Differential cloning using in‐gel competitive reassociation
Authors: Yokota Hiroshi , Amano Shinya , Yamane Tetsuo , Ataka Kazuto , Kikuya Eriko , Oishi Michio 
Published: May 2005

AbstractWe describe the principle and actual processes of a differential cloning procedure designed for cloning of anonymous restriction DNA fragments whose molecular sizes differ between two genomic DNA preparations from higher organisms as a result of DNA rearrangement, polymorphism, etc. The procedure, which was extensively modified from the original one and still employs in‐gel competitive reassociation (IGCR) as the basic principle, aims for cloning of DNA fragments which exist in one copy or less per mammalian genome. The modified procedure consists of dissociation and reassociation of biotinylated restriction digests of target DNA fragments (from which clones are to be isolated) in the presence of a large excess of reference (competitor) DNA in gel after electrophoresis, which is followed by absorption of the target DNA fragments to streptavidin‐coated tubes and solid‐phase polymerase chain reaction. After repeating these steps we attained substantial enrichment of altered DNA fragments which were originally present in one copy or less per complex mammalian genome.
Rapid chemiluminescent detection of mRNAs on Northern blots with digoxigenin end‐labelled oligonucleotides
Authors: Trayhurn Paul , Duncan Jacqueline S. , Nestor Antoinette , Thomas Moira E. A. , Eastmond Nigel C. , Rayner D. Vernon 
Published: May 2005

AbstractA simplified, nonradioactive procedure for the detection of specific mRNAs on Northern blots has been developed, utilizing digoxigenin‐labelled oligonucleotides and chemiluminescence. Antisense oligonucleotide (30–35 mer) probes were designed and synthesised based on published cDNA and gene sequences. These probes were end‐labelled (5′) with digoxigenin. Total RNA was fractionated by agarose gel electrophoresis and capillary blotted onto positively charged nylon membranes. After hybridization, the mRNA/digoxigenin‐labelled oligonucleotide complex was detected by a chemiluminescence‐based method using disodium 3‐(4‐methoxyspiro‐[1,2‐dioxetane‐3‐2′(5′chloro)‐tricyclo[3.3.1.13.7]decane]‐4‐yl)phenyl phosphate (CSPD) as substrate. The advantages of this simplified technique for detecting mRNAs in physiological and nutritional studies are described.
Destaining of Coomassie Brilliant Blue R‐250‐stained polyacrylamide gels with sodium chloride solutions
Authors: Sreeramulu Guttapadu , Singh Nagendra K. 
Published: May 2005

AbstractA novel method for destaining of polyacrylamide gels, stained with Coomassie Brilliant Blue R‐250, is described, based on the use of 0.5 M NaCl in water as the destainer, requiring only 2–3 h. Concentrated (> 2 M) or dilute (< 0.1 M) salt solutions were unsuitable. The method affords the advantage that expensive organic solvents, such as methanol, acetic acid or trichloroacetic acid, are not needed. Furthermore, salt destaining results in darker purple‐blue protein bands as compared to the pale blue color with e.g. methanol/acetic acid destaining.
Studies on the procedure for the construction of cellular protein databases employing micro two‐dimensional electrophoresis: An HL‐60 protein database
Authors: Manabe Takashi , Yamamoto Hideko , Kawai Makoto 
Published: May 2005

AbstractThe procedure to construct a cellular protein database employing a micro two‐dimensional electrophoresis (2‐DE) technique was studied. Protein information on human cultured leukemic cell HL‐60 was collected as an HL‐60 protein database, which consists of a 2‐DE polypeptide map of 670 recognized spots with spot numbering and information on isoelectric point (pI), molecular mass, and percent integrated density. The procedure employed in this report will be applicable for the comparative studies of cellular proteins and will help to construct comprehensive cellular protein databases.
Selective preconcentration for capillary zone electrophoresis using protein G immunoaffinity capillary chromatography
Authors: Cole Laura J , Kennedy Robert T. 
Published: May 2005

AbstractCapillaries with 150 μm inner diameter were packed with a perfused protein G chromatographic support and used as immunoaffinity preconcentrators for capillary zone electrophoresis. Antibody was loaded onto the protein G support to form an immunoaffinity stationary phase. Injection of samples onto the column caused selective retention and preconcentration of antigen. Injection of appropriate buffers onto the column caused desorption of the antibody and antigen which were then separated by capillary zone electrophoresis. The combination was used on‐line and off‐line. For on‐line combination, a flow‐gated interface coupled the two columns and allowed injection of desorbed zones onto the electrophoresis system. Off‐line coupling required collection of desorbed fractions and then injection onto the electrophoresis system. Flow rates as high as 100 μL/min were used to load sample onto the affinity column. Desorbing flow rates had to be 1 μL/min or less to prevent excessive dilution during desorption. Using the system, 1 mL insulin samples could be loaded onto the affinity column and desorbed in volumes as small as 1 μL for 1000‐fold preconcentration. The use of the preconcentrator with serum samples spiked with insulin was demonstrated.
Charge and size effects in the capillary zone electrophoresis of nuclease A and its variants
Authors: Kálmán Franka , Ma Stacey , Hodel Alec , Fox Robert O. , Horváth Csaba 
Published: May 2005

AbstractThe migration behavior of nuclease A from Staphylococcus aureus and 11 of its variants in capillary zone electrophoresis (CZE) was investigated in the light of their three‐dimensional structure known from X‐ray crystallography and nuclear magnetic resonance (NMR) measurements. Nuclease A (molecular mass 16.8 kDa, pKa 10.3) and the variants differ only in a single amino acid residue and have a very similar crystal structure. With the use of coated quartz capillaries and suitable buffers, the protein migration was investigated at pH from 2.8 to 9.5 without interference by wall adsorption. Although the selectivity of the electrophoretic system for the proteins was mainly determined by their charge differences, certain variants having the same net charge could also be readily separated under nondenaturing conditions. For instance, the mobility of variant K116A was sufficiently higher than that of K116G so that they could be separated by CZE. The structures of both variants are the same except for the solvent‐exposed loop containing residue 116. For this reason, the difference in electrophoretic mobilities can be attributed to the fact that in K116G the backbone of the 112 to 117 amino acids protrudes slightly from the protein, with a concomitant increase in the hydrodynamic radius with respect to that of K116A. Consequently, K116G shows a smaller mobility than K116A due to its larger hydrodynamic radius despite its smaller molecular mass. The interpretation of the experimentally measured mobilities of such closely related proteins therefore requires not only consideration of their electrostatic charge but also the fine details of their molecular structures.
Zinc‐imidazole positive: A new method for DNA detection after electrophoresis on agarose gels not interfering with DNA biological integrity
Authors: Hardy Eugenio , Sosa Angela E. , Pupo Elder , Casalvilla Racmar , Fernandez‐Patron Carlos 
Published: May 2005

AbstractNucleic acids separated by gel electrophoresis are commonly detected within the gel matrix with ethidium bromide staining, followed by gel irradiation with ultraviolet (UV) light. When the separated nucleic acids are to be recovered for further characterization or use, this methodology is unsuitable (i) because a significant number of chemical lesions to the nucleic acid molecules are caused, heavily compromising their biological a ctivity, and (ii) because of health hazards due to accumulative direct contact with ethidium bromide and exposure to UV‐light. As an alternative, for preparative purposes, a new non‐toxic detection method employing zinc and imidazole salts is described. After electrophoresis, the gel is first washed in distilled water to substantially remove remaining electrophoresis reagents, then incubated in 40 mM zinc sulfate for 10 min to allow binding of Zn2+ to the DNA, and subsequently washed with distilled water to remove unbound Zn2+ from gel regions devoid of DNA. On soaking in 0.2 M imidazole for a few minutes, zinc‐DNA complexes are visualized as deep‐white (positive) stained bands against a slightly opaque background. The sensitivity is similar to that of ethidium bromide. Gels can be kept in distilled water for months without loss of staining. After zinc chelation, e.g. with EDTA, it is feasible to quantitatively recover chemically intact and biologically active DNA from the gels, as shown by reelectrophoresis and transformation experiments.
Construction of an internal standard for semi‐quantitative polymerase chain reaction analysis of heat shock proteins
Authors: Khan Mahboob A. , Latif Najma , Petrobelli Paola , Yacoub Magdi H. , Dunn Michael J. 
Published: May 2005

AbstractWe describe a simple and effective method for generating competitive cDNA fragments for use as an internal standard in semi‐quantitative polymerase chain reaction (PCR). The use of a nested composite primer to a region slightly within the expected amplicon but with heat shock protein 60 kDa (hsp60) primer sequence appended to the 5′ terminus of the primer produces a “mimic” DNA that will model the PCR kinetics of the target template in sequence context, and PCR primer site, and that will be similar in size. This ensures that both competitor and target template are subjected to similar PCR kinetics and so allows more meaningful quantitation.
pH gradient electrophoresis of basic ribonucleases in sealed slab polyacrylamide gels: Detection and inhibition of enzyme activity in the gel
Authors: Nadano Daita , Yasuda Toshihiro , Sawazaki Kazumi , Takeshita Haruo , Kishi Koichiro 
Published: May 2005

AbstractA simple method for the separation and specific detection of basic ribonucleases (RNases) was developed. The separation was achieved by polyacrylamide gel electrophoresis in a pH gradient generated by a carrier ampholyte (Pharmalyte 8–10.5) and arginine. In order to prevent interference from atmospheric carbon dioxide, the pH gradient was formed in sealed vertical gel slab. Human nonsecretory‐type RNase, bovine pancreatic RNase A, and other basic proteins could be resolved without expensive equipment or complicated procedures. For activity detection after electrophoresis a zymogram technique was applied, using dry agarose film containing ethidium bromide plus RNA as substrate. This approach affords two advantages: (i) Basic RNase activities can be detected within 15 min, even in crude materials. The sensitivity is better than 0.5 ng of purified human nonsecretory‐type RNase. (ii) An inhibition test of RNase activities in the gel, using human placental‐type RNase inhibitor, can be performed.
Preparative electrophoresis on linear polyacrylamide‐agarose composite gels
Authors: Shainoff John R. , Smejkal Gray B. , Mitkevich Olga , Dibello Patricia M. 
Published: May 2005

AbstractA preparative method for isolating centigram quantities of high molecular weight polypeptide chains with high resolution and recovery uses linear polyacrylamide/agarose composite (LPAC) gels as electrophoretic media from which the polypeptides can be easily extracted. The composites are prepared in a manner yielding linear copolymers of acrylamide and 1‐allyloxy‐2,3‐propanediol within 2% agarose gels. After electrophoresis in sodium dodecyl sulfate (SDS), protein bands were rapidly visualized for excision by briefly immersing the gel in cold 0.1 M KCl which precipitates the protein‐associated SDS. The gel slices are then freeze‐thawed to disrupt the agarose matrix and promote syneresis of fluid upon centrifugation. The polypeptides are then separated from the polycarylamide in the supernatant solution by precipitating with either acidic isopropanol, trichloroacetic acid, ammonium sulfate or other general protein precipitants. As determined with polypeptide chains of fibrinogen and its cross‐linked derivatives, recoveries were virtually complete (95.4% ± 2.2%), and were independent of molecular weights over the range tested (104–106).
Biochemical changes during regeneration of sunflower (Helianthus annuus L.)
Authors: Martinelli Lucia , Gianazza Elisabetta 
Published: May 2005

AbstractThe main developmental stages in Helianthus annuus organogenesis have been studied in the sunflower hybrid “Giove”. Shoot regeneration was obtained with high efficiency from mature seed cotyledons. Two‐dimensional electrophoresis of protein extracts as well as the isozyme patterns of acid phosphatase, alcohol dehydrogenase, esterase, gluconate‐6‐phosphate dehydrogenase and phosphoglucomutase were compared during growth, callusing and regeneration. Two‐dimensional protein patterns were similar, although polypeptides specific for each developmental phase could be identified. Different 2,4‐dichlorophenoxyacetic acid concentrations or the sampling of specific regions of the seed did not result in significant differences in protein patterns. The activity of alcohol dehydrogenase and phosphoglucomutase appeared very low. For gluconate‐6‐phosphate dehydrogenase no difference, related either to the genotype or to different morphological stages, could be observed; the expression of acid phosphatase varied in a nonsystematic fashion. The isozyme pattern of esterase was related to the genotype as well as to the morphogenic phase.
Preparative affinity membrane electrophoresis
Authors: Horvath Z. Stephen , Gooley Andrew A. , Wrigley Colin W. , Margolis Joel , Williams Keith L. 
Published: May 2005

AbstractUsing the patented Gradiflow system in conjunction with newly developed affinity membranes, the suitability of an electrokinetic technique for affinity fractionation was investigated. Blue dextran incorporated into an affinity membrane was used to deplete a solution of horse serum of albumin, with the result that a majority of serum proteins were enriched tenfold relative to albumin. The technique, when fully developed, would offer some advantages over affinity chromatography, since to a degree it is possible to control which components of the sample are presented to the affinity matrix. Furthermore, the technique would extend the capabilities of the already multifunctional Gradiflow system.
DNA profiling and plant variety registration: 1. The use of random amplified DNA polymorphisms to discriminate between varieties of oilseed rape
Authors: Lee David , Reeves James C. , Cooke Robert J. 
Published: May 2005

AbstractBefore they can be marketed in the UK, newly bred varieties of crop species have to undergo a process of statutory testing, part of which involves the examination of the distinctness, uniformity and stability (DUS) of the variety. DUS testing is also used as the basis for the award of Plant Breeders' Rights. This paper examines the potential of DNA polymorphisms, amplified using arbitrary primers (RAPDs) for use in DUS testing of varieties of oilseed rape. RAPDs using suitable primers can produce high levels of discrimination (> 95%) between varieties, although there are certain problems in gel ‘scoring’ that are only partially resolved by computerised gel scanning/evaluation techniques. Varieties of oilseed rape are also heterogeneous in their RAPD profiles using certain primers, which could cause problems in the DUS testing context. DNA profiling with RAPDs could be used for discrimination between and identification of oilseed rape varieties, but its use for DUS testing needs to be considered carefully.
Fingerprinting of glycans as their 2‐aminoacridone derivatives by capillary electrophoresis and laserinduced fluorescence
Authors: Harland Gary B. , Okafo George , Matejtschuk Paul , Sellick Ian C. , Chapman George E. , Camilleri Patrick 
Published: May 2005

AbstractCapillary electrophoresis and laser‐induced fluorescence detection have been used to fingerprint the 2‐aminoacridone derivatives of complex glycans released from bovine fetuin and human IgG monoclonal antibodies. The utility of this method in distinguishing between N‐ and O‐linked oligosaccharides and in determining the presence of sialic acid residues in glycan mixtures at an early stage of analysis has been demonstrated.
Electrophoretic separation of recombinant tissue‐type plasminogen activator glycoforms: Validation issues for capillary isoelectric focusing methods
Authors: Moorhouse Kathryn G. , Rickel Carina A. , Chen Anthony B. 
Published: May 2005

AbstractAttempts were made to validate a capillary isoelectric focusing (cIEF) method for a recombinant glycoprotein as an alternative technique to slab gel isoelectric focusing methods routinely used to monitor such charge heterogeneity. The cIEF method principally separates the charged glycoforms of recombinant tissue‐type plasminogen activator (rt‐PA) on the basis of their sialic acid content. Nine to ten distinct peaks were consistently resolved, with the profile dependent on the class of ampholyte used. The pI of rt‐PA measured with synthetic pI standards was in the range pH 6.5–7.5 with the migration of the standards affected by the presence of the protein. The method showed an acceptable recovery of > 100% and had good sensitivity where 25 ng of protein could be resolved into constituent peaks. Recovery of both major peaks and total protein measured by peak areas was linear over a wide range from 50–1000 μ/mL. A detailed study showed that when a capillary had been used for some time, capillary age affected peak migration times and, to a lesser extent, resolution. Peak migration times were stable over a temperature range of 15–30°C, and decreased predictably with increasing voltages (400–600 V/cm) and decreasing N, N, N′, N′‐tetramethylethylene diamine (TEMED) concentrations (0.4–1.5% v/v). Overall the data indicated that this methodology has the potential to be used in the commercial release of protein pharmaceuticals if variability resulting from capillary age and lot were resolved. Even in its present format the method equals the performance of slab gel IEF whilst offering significant improvements in ease of operation and in time and reagent use.
State of the art of large‐scale genetic purity testing of hybrid vegetable seeds using isoelectric focusing at PetoSluis
Authors: Burg Henk C. J. , van den Berg Bartel M. 
Published: May 2005

AbstractDuring the past ten years we have been engaged in developing and applying isoelectric focusing techniques to test the genetic quality of vegetable seeds. We started with isoelectric focusing using carrier ampholytes (IEF‐CA), and continued research with isoelectric focusing in immobilized pH gradients (IEF‐IPG) and two‐dimensional electrophoresis (IPG‐DALT). In addition, we have developed equipment and procedures for large‐scale seed and seedling homogenization, sample preparation and semi‐automatic gel staining. Moreover, we have optimized the sample application and gel running setup for large‐scale analysis. We have developed hybrid purity (inbred) testing methods for all important vegetables, e.g., melon, cole crops, tomato, pepper, watermelon, squash, cucumber, radish etc. using either IEF‐CA or IEF‐IPG of seed or seedling proteins, followed by specific or general protein staining. To indicate the efficiency of the equipment and procedures developed we present results of two of our hybrid purity test methods, namely for brassica using polymorphism for phosphoglucomutase (PGM) from dry seeds, and for tomato using polymorphism for alcohol dehydrogenase (ADH) from imbibed seeds. We show that one person can routinely analyze 1536 individual seeds per day at a cost of about US $0.11 per seed for chemicals, materials, and electrophoresis equipment.
Quantitation of polymerase chain reaction‐amplified DNA fragments by capillary electrophoresis and laserinduced fluorescence detection
Authors: Vincent Ursula , Patra Guy , Therasse Joël , Gareil Pierre 
Published: May 2005

AbstractQuantitation of DNA fragments amplified by polymerase chain reaction (PCR) is needed for the determination of target DNA in molecular biology. Capillary electrophoresis in entangled polymer solution coupled to laser‐induced fluorescence detection was assessed as an alternative technique to conventional slab gel methods to monitor competitive PCR, which consists of amplifying an internal standard fragment under the same conditions as the target fragment. The fluorescence signal was generated either through end‐labeling of the fragments using 5′‐fluorescein‐labeled primers or through intercalation of ethidium bromide along the double strand. It is shown that the more accurate and reliable results were obtained using this latter pathway.
Subtyping of group‐specific component and protease inhibitor by rapid isoelectric focusing on PhastSystem
Authors: Yaojun Guo , Xinhui Li , Lili Huang 
Published: May 2005

AbstractA rapid method on PhastSystem was used to investigate the distribution of group‐specific component (GC) and protease inhibitor (PI) subtypes and their gene frequencies from 190 unrelated healthy donors of the Han population in Beijing. Laboratory‐made gels (pH 4.5–5.4 and pH 4.2–4.9) were used for analysis of GC and PI, respectively. Sample loading was 1.5 μL. The separation and visualization time was 0.5 h in each. Gene frequencies were as follows: GC*1F = 0.4891, GC*1S = 0.2432, GC*2 = 0.2678; rare GC variants were discovered in seven cases. The results for PI were; PI*M1=0.7542, PI*M2 = 0.1808, PI*M3 = 0.0650. Good agreement between the observed and expected values in both GC and PI subtyping (for GC, ∑χ2= 1.4043, 0.7<P< 0.8; for PI, ∑χ2= 1.1233, 0.7 <P< 0.8) was obtained.