bluuebunny/crossref_metadata_2025_split huggingface dataset:
Detection of exo‐β‐1,3‐glucanase activity in polyacrylamide gels after electrophoresis under denaturing or nondenaturing conditions
Authors: Vargić Tamara , Mrša Vladimir 
Published: May 2005

AbstractA method for the visualization of exo‐β‐1,3‐glucanase activity in polyacrylamide gels is presented. The procedure consists of the enzyme reaction in the gel with the substrate α‐naphthylglucopyranoside, and a subsequent staining of the obtained α‐naphthol with dyes Fast Red B, or Fast Blue BB, respectively. A mixture of exoglucanases produced by the fungus Polyporus squamosus was used for the optimization of the method. The procedure is applicable for the standard Laemmli discontinuous electrophoresis system, even in the presence of sodium dodecyl sulfate, as well as for electrophoresis in linear gradients of the polyacrylamide concentration. The staining method was used for the analysis of exoglucanases secreted by several yeast genera. All yeasts tested produced two types of exoglucanases, a high molecular mass species heterogeneous in size, and one or two smaller homogeneous enzymes.
Analysis of mephenytoin, 4‐hydroxymephenytoin and 4‐hydroxyphenytoin enantiomers in human urine by cyclodextrin micellar electrokinetic capillary chromatography: Simple determination of a hydroxylation polymorphism in man
Authors: Desiderio Claudia , Fanali Salvatore , Küpfer Adrian , Thormann Wolfgang 
Published: May 2005

AbstractUsing cyclodextrin micellar electrokinetic capillary chromatography (CD‐MECC), baseline separation of mephenytoin, 4‐hydroxymephenytoin and 4‐hydroxyphenytoin enantiomers in urine was effected with β‐cyclodextrin. After single‐dose administration of 100 mg of racemic mephenytoin, the 0–8 h urine was collected, and enzymatically hydrolyzed urine specimens were applied. For extensive metabolizers, a single peak for 4‐hydroxymephenytoin was detected corresponding to the S‐enantiomer. This peak was either very small or undectable in samples of poor metabolizers. Typically, mephenytoin could not be detected in these samples. However, application of undeglucu‐ronidated extracts revealed the presence of free S‐4‐hydroxymephenytoin and R,S‐mephenytoin and thus permitted phenotyping via both the urinary S:R enantiomeric ratio of mephenytoin and the hydroxylated metabolite. Application of enzymatically hydrolyzed and extracted urines after phenytoin administration (100 mg; 0–8 h urine collection) revealed the presence of S‐4‐hydroxyphenytoin. Thus, CD‐ MECC is shown to be a simple and attractive approach for (i) the confirmation of the stereoselectivity of the aromatic hydroxylation of mephenytoin and phenytoin, (ii) the simple and rapid differentiation between extensive and poor metabolizers for mephenytoin, and (iii) assessment of compliance.
Renaturation and activity staining of glycosidases and glycosyltransferases in gels after sodium dodecyl sulfate‐electrophoresis
Authors: Mukasa Hidehiko , Tsumori Hideaki , Takeda Hiroyuki 
Published: May 2005

AbstractGlycosidases and glycosyltransferases were electrophoresed in the presence of sodium dodecyl sulfate (SDS) in a thin‐layer gel supported by a glass plate, treated with the nonionic detergent Triton X‐100, and specifically stained for the sugar‐releasing activity of these enzymes. Staining is based on conversion of monosugars or a sugar phosphate to glucose‐6‐phosphate by the appropriate intermediary enzymes, reduction of NADP+ to NADPH, and accumulation of reduced Nitroblue Tetrazolium in the gel. Among the enzymes tested, α‐glucosidase, β‐glucosidase and β‐mannosidase could not be renatured, whereas β‐fructofuranosidase and α‐mannosidase could be renatured unless heated before electrophoresis. Sucrose phosphorylase, glucosyltransferase and fructosyltransferase, which are single‐peptide proteins with no cystine bond, could be renatured even after pretreatment with SDS and/or mercaptoethanol at 100°C for 10 min. However, exclusive heating remarkably decreased the activities of these enzymes. Two‐dimensional separation of the five renaturable enzymes was done in a single thin‐layer gel, using SDS‐electrophoresis in the first dimension and isoelectric focusing in the second dimension.
Quantitation of electrophoretically separated proteins in the submicrogram range by dye elution
Authors: Neumann Ulf , Khalaf Hosni , Rimpler Manfred 
Published: May 2005

AbstractA new method for the fast, reliable and reproducible submicrogram quantitation of proteins separated by different electrophoretic techniques is presented. The method is based on a modified sensitive staining technique using Coomassie Brilliant Blue G‐250 in colloidal solution combined with an optimized elution procedure of the bound dye in a 3% w/v solution of sodium dodecyl sulfate followed by photometric determination of dye concentration in the eluate. In addition a new method is provided for background correction, even suitable for gels showing strong background staining. The staining procedure allows the detection of 20 ng depending on the nature of the protein and the separation technique used. Quantitation is linear at least in the range from 50 ng to 10 μg and highly reproducible even under non‐optimized conditions. The presented method can be applied to sodium dodecyl sulfate‐electrophoresis, isoelectric focusing and two‐dimensional electrophoresis.Part of the thesis of Ulf Neumann, Medizinische Hochschule Hannover 1992.
Filipin staining of lipoproteins in polyacrylamide gels: Sensitivity and photobleaching of the fluorophore and its use in a double staining method
Authors: Smejkal Gary B. , Hoff Henry F. 
Published: May 2005

AbstractWe have developed a double staining procedure in which polyacrylamide gels are first stained with filipin to identify lipoproteins, and then with Coomassie Brilliant Blue (CBB) to identify proteins. Filipin staining when performed at 37°C is both more rapid and more sensitive than previously published procedures. After only 5 min, 20 ng of low density lipoprotein (LDL) unesterified cholesterol/mm3 of band volume could be detected, and after 12 h, sensitivity reached 0.8 ng/mm3. A semilogarithmic relationship was found between the amount of LDL unesterified cholesterol applied and filipin fluorescence. Although rapid photobleaching of the fluorophore occurred during UV transillumination of these gels, such photobleaching actually resulted in maximizing of the signal:noise ratio, resulting in better definition of bands. Treatment of gels with filipin had no deleterious effects on the subsequent staining with CBB. This dual staining procedure should prove useful for studies in which both lipoproteins and proteins in plasma need to be documented in the same gel.
Method for rapid electrophoretic transfer of isoelectric focusing gels to polyvinylidene difluoride
Authors: Lobert Sharon , Correia John J. 
Published: May 2005

AbstractWe present here a protocol for unidirectional wet electrotransfer of isoelectric focusing (IEF) gels to polyvinylidene difluoride (PVDF) that can be carried out in < 2 h, including preequilibration steps. In developing this protocol, we systematically evaluated sodium dodecyl sulfate and methanol concentrations in the preequilibration washes and length of electrotransfer. We demonstrated that the method is useful for tubulin, a protein that resists usual semi‐dry transfer procedures due to its limited solubility under IEF conditions. We also successfully used our method for transferring the microtubule‐associated protein, tau, as well as carbamylyte standards, creatine phosphokinase and carbonic anhydrase.
Quantitative analysis of hyaluronan in vitreous humor using capillary electrophoresis
Authors: Grimshaw James , Kane Áine , Trocha‐Grimshaw Jadwiga , Douglas Alistair , Chakravarthy Usha , Archer Desmond 
Published: May 2005

AbstractCapillary electrophoresis was used for the separation and quantitative analysis of the glycosaminoglycan hyaluronan in human and bovine vitreous with detection by UV absorbance at 200 nm. Calibration was carried out using standards made up from known concentrations of hyaluronan of umbilical cord origin. The purity of the standard was examined by1H NMR (nuclear magnetic resonance). Concentrations as low as 25 μg/mL could be detected by capillary electrophoresis. Confirmation that the signal was due to hyaluronan was obtained by depolymerization of the native mucopolysaccharide by hyaluronidase. This resulted in loss of the hyaluronan peak and appearance of several new peaks corresponding to the oligomeric fragments which had shorter migration times. Capillary electrophoresis is a reproducible and sensitive technique for the quatification and characterisation of hyaluronan in vitreous samples.
Differentation by preparative continuous free flowisoelectric focusing of cyclosporin A inhibitable peptidyl‐prolyl cis/trans isomerase of human erythrocytes
Authors: Küllertz Gerhard , Meyer Sylke , Fischer Gunter 
Published: May 2005

AbstractPreparative continuous free flow‐isoelectric focusing has been used to separate at least three different components of intrinsic peptidyl‐prolyl cis/trans isomerase (PPIase) activity from erythrocytes lysate. By adding chemical spacer molecules like glycine and Bicine to commercial carrier ampholyte mixtures the resulting pH profile was predictably influenced. With an applied field strength of 125–170 V/cm a residence time of less than 15 min was sufficient for the separation of PPIases with isoelectric points of 5.4, 5.7 and 5.9 from the bulky hemoglobin. The recovery of the overall PPIase activities was about 100%. The purification factor has been determined as 20‐ to 100‐fold. For each isoform of the enzyme the peptidyl‐prolyl cis/trans isomerase acitivity of the separated proteins was inhibited by cyclosporin A but was resistant toward FK 506.
Analysis on heterogeneity of squamous cell carcinoma antigen by two‐dimensional electrophoresis
Authors: Abe Hiroaki , Okuno Nagato , Takeda Osamu , Suminami Yoshinori , Kato Hiroshi , Nakamura Kazuyuki 
Published: May 2005

AbstractSquamous cell carcinoma (SCC) antigen was separated by two‐dimensional electrophoresis combined with immunoblotting into four spots: spot 1 with pI 6.4 and 44.5 kDa, spot 2 with pI 6.3 and 44.5 kDa, spot 3 with pI 6.0 and 44.5 kDa, and spot 4 with pI 5.9 and 45 kDa. In cancer and noncanerous tissues, it was common that spot 1 was the largest spot. In noncancerous tissues, spot 3 was the smallest spot and spot 2 was stained as densely as spot 4. In cancer tissues, however, spot 4 was apparently smaller than spot 2 and 3. Also, spots 2 and 3 in cancer tissues were larger than those in noncancerous tissues. When SCC antigen was treated with alkaline phosphatase prior to isoelectric focusing (IEF), spot 4 disappeared from the immunoblotting pattern. When the SCC antigen was treated with alkaline phosphatase after IEF, spot 4 changed its molecular weight to the same weight as that of the other three spots. These results strongly suggest that spot 4 is phosphorylated SCC antigen.
Immobilized buffers for isoelectric focusing: From gradient gels to membranes
Authors: Righetti Pier Giorgio , Giaffreda Emilio 
Published: May 2005

AbstractSome major improvements to a pH gradient simulator for isoelectric focusing in immobilized buffers are here described. They allow creation of graphs (giving the profiles of pH gradient, deviation from ideal shape, buffering power and ionic strength) with up to 200 increments (which, in a 1 pH unit span means mapping of pH intervals at barely 0.005 pH increments per step).Computer program now available from Hoefer Scientific Instruments (654 Minnesota Street, San Francisco, CA 94107, USA).
In addition, for preparative purification protocols, utilizing multicompartment electrolyzers with isoelectric, buffering membranes, this modified program allows easy calculation of the precise pI value at any point along the pH gradient used for the analytical assessment of the pI values of the proteins to be purified. At the desired pH value, reached by moving the cursor along the pH gradient, a simple clicking of the mouse button allows the display of the instant Immobiline composition responsible for that pH value. The molarity ratio of the various Immobilines present at this precise point of the pH scale in fact gives the composition (with the accompanying value of buffering power and ionic strength) of the membrane to be utilized as a pH‐stat in the preparative scale run. This is the only method that allows reproducible and valid scaling up from the analytical to the preparative scale.
Separation of chlorophyll‐protein complexes by deriphat polyacrylamide gradient gel electrophoresis
Authors: Sárvári Éva , Nyitrai Péter 
Published: May 2005

AbstractAn improved Deriphat polyacrylamide gradient gel electrophoresis system was developed for the separation of chlorophyll‐protein complexes. The relatively good resolution of the starting discontinuous gel system was further improved by using glycerol in gels and an acrylamide gradient with high acrylamide‐to‐N,N′,‐methylenebisacrylamide ratio in the separating gel. By applying mild but efficient glycosidic detergents for solubilization, and Deriphat to gels and buffers, the stability of complexes was increased, and only a low amount of pigment was removed. The advantage of our system is the better resolution of larger‐size complexes, especially those of photosystem I. In addition, it makes possible an easier interpretation of results due to less overlapping of photosystem I and photosystem II bands when different plant species or the effects of different treatments are compared using whole thylakoid membranes.
Separation of large DNA molecules with high voltage pulsed field gel electrophoresis
Authors: Wagner Lauren , Lai Eric 
Published: May 2005

AbstractWe have developed two high voltage (3 kV and 10 kV) high speed pulsed field gel electrophoresis systems for the separation of DNA as large as 460 kbp. These systems enable us to combine the rapid speed of high voltages and the separation power of pulsed field electrophoresis to achieve high resolution and short run durations. We found that large DNA fragments can be separated at voltage gradients much higher than commonly used. Yeast chromosomes as large as 460 kbp can be separated in 4 h at 20 V/cm and 1–50 kbp DNA can be rapidly separated in 30 min at 55 V/cm. This is 25 times faster in mobility for the separation of relatively small DNA fragments (< 50 kbp). We have also found an inverse relationship between the voltage applied and the size separation limit at that particular voltage gradient (55 V/cm limits the separation to 50 kbp while 20 V/cm can separate up to 460 kbp). Depending on the size range, DNA can be separated 8‐ to 25‐fold faster and with better resolution than existing electrophoretic systems.
The relative separation efficiencies of highly concentrated, uncrosslinked or low‐crosslinked polyacrylamide gels compared to conventional gels of moderate concentration and crosslinking
Authors: Zakharov Sergey F. , Chrambach Andreas 
Published: May 2005

AbstractThe joint report [1] has shown that the separation of heteroduplex DNA from homoduplex DNA can be achieved by uncrosslinked polyacrylamide gels or gels of a very low degree of crosslinking (0.15%) with N,N1‐methylenebisacrylamide (Bis), while conventional polyacrylamide gels of 2–5% crosslinking with Bis are incapable of such a separation in the absence of added denaturing agents. This result raised the question whether in application to other separation problems the same superiority of uncrosslinked or low‐crosslinked polyacrylamide existed. To test that question, Ferguson plots were determined for the members of a DNA ladder (50 to 1000 bp) in polyacrylamide with 0, 0.1, 0.2, 0.3, 0.5 %C (Bis), and the separation efficiency function, S, was evaluated in comparison with that in conventional 2–5%C (Bis) gels. S was found to be lower, not higher, in gels of low crosslinking at the respective maximally effective gel concentrations. However, the range of gel concentrations in which gels of low or no crosslinking were effective extended over a range of at least 10%T, while conventionally crosslinked gels were most effective over a range of 3 to 1 units of %T.
Focused antigen capture immunoassay
Authors: Suen Kitty K. Y. , Lewis W. H. P. 
Published: May 2005

AbstractA new approach is described to immunoblotting in which antigens after isoelectric focusing are captured on nitrocellulose membranes coated with a specific antibody. The method has been applied to the analysis of human IgA ϰ and λ from whole blood and serum without prior purification. The visualized bands were quantified by laser densitometry in four pI ranges. It is concluded that, under the conditions used, close to 100% of serum IgA is captured. The method has a coefficient of variation of 4.5–12.8%, mean 8.0%.
Electrophoretic mobilities of the complexes between sodium dodecyl sulfate and various peptides or proteins determined by free solution electrophoresis using coated capillaries
Authors: Karim Mohammad R. , Shinagawa Susumu , Takagi Toshio 
Published: May 2005

AbstractElectrophoretic mobilities of various proteins or peptides complexed with sodium dodecyl sulfate (SDS) were determined by free solution capillary electrophoresis. The complexes formed between SDS and protein polypeptide showed electrophoretic mobilities virtually insensitive to the protein molecular weight. On the other hand, scattered and larger negative electrophoretic mobilities were observed for peptides of molecular weights less than 10000. Mobility differences as small as 0.3–3% among the three differently modified derivatives of bovine serum albumin could also be successfully determined due to the high resolving power of capillary electrophoresis.
Separation of cis/trans isomers of a prolyl peptide bond by capillary zone electrophoresis
Authors: Meyer Sylke , Jabs Andreas , Schutkowski Mike , Fischer Gunter 
Published: May 2005

AbstractOn capillary electrophoresis of the chemically pure thioxo peptide Ala‐Phe‐ψ[CS‐N]‐Pro‐Phe‐4‐nitroanilide a peak splitting was observed at a capillary temperature of 25°C. By contrast, the oxo peptide analogue exhibits a single, sharp peak under these conditions. Both peaks of the thioxo compound coincided gradually when the temperature was increased to 60°C. Peak fusion was reverted by cooling down the heated sample. This behavior could be attributed to the electrophoresis‐mediated separation of the cis/trans prolyl bond isomers of the thioxo peptide, allowing data of this conformational equilibrium to be determined. Derived from computational data about molecular volume and the hydration energy of low‐energy cis and trans isomeric structures, the more rapid migration of the cis form in comparison to trans may be explained by structural parameters.
Molecular weight alterations of alpha‐1 proteinase inhibitor in equine bronchoalveolar lavage fluid
Authors: Milne Elspeth M. , Pemberton Alan D. , McGorum Bruce C. , Dixon Padraic M. , Miller Hugh R. P. 
Published: May 2005

AbstractThe equine alpha‐1 proteinase inhibitor (α1PI) system differs from that of man in that the equine system consists of four closely‐linked genes (Spil‐Spi4) whereas in man, a single gene encodes for α1PI. We have previously found differences in the proportion of the Spi proteins in equine serum and bronchoalveolar lavage fluid (BALF). We therefore wished to determine whether, as reported in man, there was any molecular weight difference between the Spi proteins in serum and BALF. α1PI and albumin from equine BALF migrated further towards the anode compared with serum α1PI on native polyacrylamide gel electrophoresis (PAGE) although the difference was only significant for α1PI. Sodium dodecy1 sulphate‐PAGE (SDS‐PAGE) showed that a mean decrease in molecular weight of 1.5 kDa for α1PI and 1.3 kDa for albumin had occurred in BALF. These findings were observed in control animals and in those with symptomatic or asymptomatic chronic obstructive pulmonary disease. The mechanism of this decrease in molecular weight of α1PI is likely to differ from reports of α1PI cleavage by bacterial proteinases in man since the molecular weight change was relatively small and loss of trypsin inhibitory activity did not occur. Nor, in our system, was there evidence of bacterial infection. Damage by endogenous proteinases or glycosidases at a site other than the reactive site may be involved but the resultant effect on the efficiency of the antiproteinase screen of the lower respiratory tract is uncertain.
Micellar electrokinetic chromatography with electroosmotic step gradient elution
Authors: Tsai Pei , Patel Bhisma , Lee Cheng S. 
Published: May 2005

AbstractThe peak capacity, defined as the maximum number of resolvable peaks, is dependent on the range of the migration‐time window in micellar electrokinetic chromatography (MEKC). By applying various radial electric potential gradients across the capillary wall, the direct control of the ζ potential and the electroosmotic flow in MEKC results in a significant increase in the migration‐time window and a great enhancement of the separation resolution. The enhancement in the separation resolution is obtained at a large expense in analysis time. To optimize both the separation efficiency and the resolution in MEKC, the dynamic control of electroosmosis and the migration‐time window during the separations is demonstrated with the use of neutral and charged phenylthiohydantoin‐amino acids (PTH‐amino acids) as a model system. A simple mathematic model which is capable of predicting migration times of analytes in electroosmotic step gradient elution MEKC is presented.
Micellar electrokinetic capillary chromatography with in situ charged micelles: 3. Evaluation of alkylglucoside surfactants as anionic butylboronate complexes
Authors: Smith Joel T. , El Rassi Ziad 
Published: May 2005

AbstractThis article represents an extension to a new approach introduced very recently by our laboratory for the control of the surface charge density as well as the hydrophobic character of micellar phases used in micellar electrokinetic capillary chromatography (MECC). The approach is based on the complexation of polyolic surfactants, e.g., alkylglucosides, with butylboronate to form in situ branched, anionic surfactants. The butylboronate can also incorporate into the micelle via its alkyl tail and acts as a “class I” organic additive that mainly modifies the micelle by decreasing the critical micellar concentration, i.e., increasing the hydrophobic character of the micelle, while exhibiting little influence on the aqueous phase. The net result is an in situ charged micellar entity whose hydrophobic character is dynamically altered. The alkylglucosidebutylboronate micellar phases yielded high separation efficiencies and proved useful in the separation of charged and neutral herbicides as well as the chiral separations of medicarpins and precursors, and dansylated D and L‐amino acids in the presence of native or modified cyclodextrin chiral selectors.
Effect of organic modifier on resolution of hydrophobic compounds by micellar electrokinetic chromatography
Authors: Kaneta Takashi , Yamashita Tetsuya , Imasaka Totaro 
Published: May 2005

AbstractThree organic modifiers, methanol, acetonitrile, and dimethylformamide, were used as organic modifiers to improve the resolution of hydrophobic compounds in micellar electrokinetic chromatography. The capacity factor decreased with increasing concentration of organic modifier. Acetonitrile and dimethylformamide were more effective modifiers to decrease the capacity factor than methanol. Maximum resolution was obtained when the capacity factor was reduced to about 2. Methanol enhanced the resolution for less hydrophobic compounds, while acetonitrile and dimethylformamide were better modifiers to improve the resolution for strongly hydrophobic compounds.