bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization and quantification of mink serum amyloid A protein using two‐dimensional electrophoresis with immobilized pH gradients
Authors: Bruun Cathrine Foyn , Sletten Knut , Husby Gunnar , Marhaug Gudmund
Published: May 2005

Using hydrophobic interaction chromatography, two‐dimensional electrophoresis with an immobilized pH gradient in the first dimension and semidry blotting, three isoforms of mink serum amyloid A protein (SAA) were characterized and studied during chronic inflammation. Compared to conventional methods that have been applied to SAA, the major advantages of the present combination of methods are: (i) use of small serum volumes, (ii) rapid extraction, (iii) high resolution, and (iv) high yield of proteins.
Use of Rotofor in two‐dimensional electrophoretic analysis: Identification of a 100 kDa monoclonal IgG heavy chain in myeloma serum
Authors: Goldfarb Marcia F.
Published: May 2005

After preparative nondenaturing isoelectric focusing in a Rotofor of 1 mL human serum which showed on two‐dimensional electrophoresis a monoclonal kappa light chain pI < 5.0, the monoclonal kappa light chain was identified in the tubes with pH 7.29 – pH 6.96, indicating that the chain was not free and was most likely part of an immunoglobulin molecule. The combined fractions of the Rotofor were concentrated back to 1 mL and aliquots were separated by two‐dimensional electrophoresis, followed by Western blotting. Enzyme immunoassay of the blot with A′ human IgG (γ chain specific) revealed a monoclonal heavy chain at a molecular size of approximately 100 kDa. Diffuse areas at 50 kDa (the attributed size of γ heavy chain), 100 kDa, and 150 kDa were stained also, but the strong, restricted (picket fence) bands indicating a monoclonal pattern were at 100 kDa. The monoclonal light chain appears to be part of a large or dimeric IgG molecule.
DNA shape and separation efficiency in polymer media: A computerized method based on electrophoretic mobility data
Authors: Tietz Dietmar , Chrambach Andreas
Published: May 2005

The computer program ELPHOFIT for evaluation of the nonlinear plots of log‐(mobility) vs. polymer concentration (Ferguson plots) in terms of molecular and polymer properties has been extended to yield a measure of the molecular sieving capacity of the polymer medium. The usefulness of the extended program, version 2.2, was exemplified by the evaluation of DNA shape and separation efficiency in solutions and gels of agarose and polyacrylamide, using previous reports in the literature as a data base. That application of the extended program yields the following results:(i) The size of migrating DNA can be compared with an equivalent sphere having the same free mobility for a particular set of experimental conditions. The decrease in size of the equivalent sphere with polymer concentration previously demonstrated for agarose solutions applies to all of these polymer media; it reveals a steep, hyperbolic decline of that radius in uncrosslinked polyacrylamide solutions in contrast to the shallow decline in the other three media. (ii) The separation efficiency of polyacrylamide gels exceeds that of uncrosslinked polyacrylamide solutions; the separation efficiency of agarose solutions for DNA smaller than 1 kb in length is higher than that of polyacrylamide solutions. Program ELPHOFIT 2.2 is available on request from the first author.
Retardation of serum albumin migration in native polyacrylamide gel electrophoresis by the incorporation of blue dextran in the gel matrix
Authors: Pemberton Alan D. , John Huw A.
Published: May 2005

Blue dextran was incorporated into the stacking layer of one‐dimensional polyacrylamide gels to prevent the migration of albumin into the resolving gel during the electrophoresis of native plasma samples. Human and horse plasma proteins normally masked by albumin were revealed by this method.
Pulsed‐field polyacrylamide gel electrophoresis: Basic phenomena and applications
Authors: Ito Takashi , Hohjoh Hirohiko , Sakaki Yoshiyuki
Published: May 2005

Pulsed‐field gel electrophoresis (PFGE) using polyacrylamide gels, termed pulsed‐field polyacrylamide gel electrophoresis (PF‐PAGE), had been developed for the effective separation of linear DNAs from circular ones [1]. The first generation PF‐PAGE employed horizontal polyacrylamide gels run in a contour‐clamped homogeneous electric field (CHEF) apparatus. The second generation system, using a vertical slab gel in a discontinuous buffer system and field inversion gel electrophoresis (FIGE), was found to be easier to handle and requires a much shorter time for separation than the previous one [2]. In this report, basic aspects of the second generation PF‐PAGE, such as the effects of a discontinuous buffer system and field inversion on the DNA migration in polyacrylamide gels, were investigated. The results indicate that the periodic inversion of electric field can broaden the resolving capability of polyacrylamide gels, enabling DNAs that otherwise fail to enter polyacrylamide gels to be resolved in such systems. Successful and possible applications of PF‐PAGE techniques are also discussed.
The 0.7 to 3.3 megabase chromosomes from Candida, Kluyveromyces, and Pichia provide accurate size standards for pulsed field gel electrophoresis
Authors: Zhang Tai Yong , Fan Jian‐Bing , Ringquist Steven , Smith Cassandra L. , Cantor Charles R.
Published: May 2005

Pulsed field gel electrophoresis was used to size intact chromosomal DNAs from Candida albicans, Kluyveromyces lactis, Pichia scolyti, and Pichia mississippiensis by optimization methods using, as size standards, concatenated bacteriophage lambda DNA, and intact and NotI digestion products of Schizosaccharomyces pombe chromosomal DNAs. These newly sized fungal DNAs can now serve as convenient and accurate size standards for DNA molecules between 0.7 and 3.3 megabases (Mb). These size standards are valid over a wide range of different electrophoretic conditions.
Influence of the agarose matrix in pulsed‐field electrophoresis
Authors: Kirkpatrick Francis H. , Dumais Maxine M. , White Hugh W. , Guiseley Kenneth B.
Published: May 2005

Properties of agarose potentially relevant to PFGE (pulsed‐field gel electrophoresis) are reviewed, and some new information is presented. Agarose polymers appear to have molecular weights in the range of 100 000 to 200 000 Da, but this is not tightly related to the effective gel strenght. Agarose has some residual charge, and hence exhibits electroendosmosis (EEO). It is possible to markedly increase the speed of separation of DNA molecules by using agarose of low EEO, especially in low ionic strenght, non‐borate buffers. This increase is especially noticeable in the relatively long experiments required for separation of large DNAs. It is also possible to increase the range of separation in a single run by use of step gradients of agarose concentration, which allows visualization of yeast chromosomes and lambda‐phage restriction fragments in the same lane. Because of the strong influence of concentration on separation, it may be useful for investigators to control water content and related variables. Our lack of knowledge of the detailed microstructure of gels may be barrier to complete understanding of PFGE.
Trace analysis of proteins by capillary zone electrophoresis with on‐column transient isotachophoretic preconcentration
Authors: Foret František , Szökő Eva , Karger Barry L.
Published: May 2005

The qualitative and quantitative aspects of transient isotachophoretic (ITP) sample preconcentration in the capillary zone electrophoretic analysis of protein samples have been demonstrated. By the proper selection of components of the background electrolyte and/or additives to the sample solution, two basic electrolyte arrangements have been employed. In the first, a typical isotachophoretic electrolyte system consisting of a leading and terminating electrolyte was used, and after focusing and preconcentration, the terminating electrolyte was replaced by the leading electrolyte, with the separation being continued in the zone electrophoretic mode. In the second, only one background electrolyte was used, containing a co‐ion with low electrophoretic mobility, and the sample was supplemented with a salt of a highly mobile co‐ion. In this case transient isotachophoretic migration of the sample ions took place at the beginning of the migration and gradually changed to the zone electrophoretic mode. Sample mixtures containing basic (positively charged) or acidic (negatively charged) proteins were examined using surface‐coated fused‐silica capillaries. For acidic proteins, bare silica was also tested. The isotachophoretic sample stacking permitted injection and preconcentration of sample volumes two to three orders of magnitude higher than usual in capillary zone electrophoresis. For example, up to 1 μL was injected into a 75 μm ID capillary. This approach afforded quantitative analysis of protein samples in the concentration range of 10−7–10−8 M, with detection limits of approximately 10−9 M. Furthermore, with constant sample volume injected, good reproducibility of migration times was obtained. Finally, the determination of trace components in the presence of a major sample component using transient ITP preconcentration has been demonstrated.
Screening and characterization of biopharmaceuticals by high‐performance capillary electrophoresis with laserinduced native fluorescence detection
Authors: Lee Thomas T. , Lillard Sheri J. , Yeung Edward S.
Published: May 2005

High‐performance capillary electrophoresis (HPCE) with laser‐induced native fluorescence (LIF) detection is used to address significant problems in the quality control of biopharmaceuticals. All of the biopharmaceuticals studied can be detected at subnanomolar levels with linear dynamic ranges of at least 3 orders of magnitude. HPCE/LIF can determine impurities in “purified” biopharmaceuticals present in amounts less than 0.01% (i.e., at 4 × 10−11 M) that of the major component. With HPCE/LIF, detection sensitivity is thus no longer a concern in the assaying of active ingredients in biopharmaceutical dosage formulations. The peptide mapping of biopharmaceuticals present at 1 × 10−7 M (or an injected limit of detection of 60 amol) is presented. Also, kinetic information on the reaction of a recombinant enzyme‐drug with its substrate present at the micromolar level has been extracted from electropherograms acquired in real‐time.
Contribution of secondary structure to DNA mobility in capillary gels
Authors: Konrad Kenneth D. , Pentoney Stephen L.
Published: May 2005

Intramolecular base pairing at the 3′ end of single‐stranded DNA molecules can cause increased electrophoretic mobility. In DNA sequencing separations this can result in compressions, where multiple oligomers differing in length by one base comigrate, which complicate sequence analysis. Using a novel approach to the application of capillary electrophoresis to DNA sequencing, incorporation of formamide into capillary gels, different separation field strengths, and external heating of the capillary are examined for their ability to resolve compressions. The identity of the 3′ terminal nucleotide has also been observed to influence oligomer mobility in cross‐linked acrylamide slab gels. We utilize automated data collection from DNA sequencing separations to show that a similar effect is also present in linear acrylamide capillary gels run at high field strengths. We also demonstrate the ability of capillary electrophoresis to separate synthetic oligos of the same length but differing in composition by one base, or of the same base composition but different sequence.
On the pH dependence of polymerization efficiency, as investigated by capillary zone electrophoresis
Authors: Caglio Silvia , Righetti Pier Giorgio
Published: May 2005

The conversion efficiency of 5 different catalyst systems for polyacrylamide gel polymerization has been assessed as a function of pH of the gelling solution, in the pH 4.0–10.0 range. The system of persulfate‐N,N,N′,N′‐tetramethylethylene‐diamine (TEMED) gives optimal incorporation of monomers only in the pH 7.0–10.0 range; at progressively acidic pH values, the conversion drops markedly until, at pH 4.0, no gelation occurs. In the system of riboflavin‐TEMED, the opposite behavior is observed: good conversions in the pH 4.0–7.0 interval (with a peak at pH 6.2), followed by progressive worsening at alkaline pH values until, at pH 10.0, no gelation occurs. An excellent catalyst system appears to be the complex comprising methylene blue, toluene‐4‐sulfinic acid and diphenyliodonium chloride, which is able to guarantee > 95% conversion in the pH 4.0–8.0 range. Lower efficiencies are obtained at pH 9.0 and 10.0 (down to 81 % conversion) but gelation still occurs in the entire pH range. Two other systems (ascorbic acid, ferrous sulfate and hydrogen peroxide in one case, and persulfate, TEMED and hydrosul‐fite, in another case) have been investigated. The former has some efficiency only at pH 4 (81 % conversion), with a rapid drop as pH increases (only 48 % incorporation at pH 6). The latter exhibits the opposite behavior: 70% efficiency at pH 4.0, increasing to 92 % conversion at pH 6.0.
Determination of human alcohol dehydrogenase and acetaldehyde dehydrogenase genotypes by single strand conformation polymorphism in discontinuous buffer electrophoresis
Authors: Walzer Claude , Turler Hans , Balant Luc , Golaz Olivier , Hochstrasser Denis F. , Monteiro Maristela , von Wartburg Jean‐Pierre
Published: May 2005

Under appropriate conditions single strand conformation polymorphism (SSCP) analysis of polymerase chain reaction (PCR) products allows the detection of single base mutations in a given DNA fragment. We adapted this method for the routine determination of allele variants of human alcohol and acetaldehyde dehydrogenase without radioisotopic labeling. After PCR amplification of the selected exon, the DNA fragments were heat‐denatured and loaded on a polyacrylamide gel containing glycerol. For electrophoresis a discontinuous buffer system was used with sulfate as leading ion and borate as trailing ion. The DNA bands were revealed by silver staining. Acrylamide concentrations, ionic strength and electrophoresis temperature were systematically investigated for each DNA fragment. The polymorphisms detected by SSCP were identical to those found by hybridization with 32P‐labeled allele‐specific oligonucleotides. This method avoids the use of radioactivity, is less expensive and simpler than the allele‐specific oligonucleotide (ASO) methodology and thus particularly suited for routine analysis.
Phenotypic change and altered protein expression in X‐ray and methylcholanthrene‐transformed C3H10T1/2 fibroblasts
Authors: He Chaoying , Merrick B. Alex , Witcher Lora L. , Patterson Rachel M. , Daluge Dennis R. , Selkirk James K.
Published: May 2005

The morphology, growth properties and cellular protein patterns from parent and two transformed C3H10T1/2 cell lines were analyzed to associated the phenotypic and protein differences with cell transformation. Transformed 10T1/2 cells were obtained by colony isolation after exposure of parent 10T1/2 cells to methylcholanthrene (MCA‐1 cell line) or X‐ray irradiation (XR‐III cell line). Compared to parent 10T1/2 and MCA‐1 cells, XR‐III cells were much smaller in size and exhibited the highest growth rate, greatest cell saturation density, increased plating efficiency and greater expression of proliferating cell nuclear antigen. MCA‐1 cells showed intermediate characteristics between parent and XR‐III cells. Among the three cell lines, only XR‐III cells showed anchorage‐independent growth in soft agar. When [35S]methionine‐labeled whole cell lysate proteins were separated by two‐dimensional polyacrylamide gel electrophoresis, computer comparison algorithms revealed a 97% similarity in protein profiles among almost 800 proteins detected from each cell line. However, comparison of proteins patterns of the transformed cell lines to that of parent 10T1/2 cells showed that 30 and 20 proteins were induced or repressed in XR‐III cells and MCA‐1 cells, respectively. Similarly, 81 and 24 proteins showed significant quantitative changes (threefold or greater) in XR‐III and MCA‐1 cells, respectively, as compared with parent 10T1/2 cell proteins. The ahchorage‐independent growth and increased proliferation properties of XR‐III cells suggest a later stage of transformation compared to MCA‐1 cells. Overall, these data suggest that gradational morphologic and phenotypic aberrancy in MCA‐1 and XR‐III cell lines from parent cells is accompanied by an increased diversity in protein expression in a limited set of proteins, some of which may play important roles in the multistep transformation process.
Polypeptide pattern of human breast epithelial cells following human chorionic gonadotropin (hCG) treatment
Authors: Ho Teh‐Yuan , Russo Jose , Russo Irma H.
Published: May 2005

Numerous attempts have made to describe the particular protein pattern of malignant cells by using high resolution two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE). The placental hormone human chorionic gonadotropin (hCG) inhibits tumor initiation and progression in experimental animals and has an inhibitory effect on the proliferation of human breast epithelial cells (HBEC) in vitro. The inhibitory effect on the immortalized HBEC MCF‐10F is accompanied by the immunocytochemical expression of inhibin α and β subunits by treated cells. With the purpose of clarifying the molecular mechanisms involved in this effect, the pattern of protein synthesis and mRNA were studied by 2‐D PAGE in the immortalized HBEC MCF‐10F cells teated in vitro with 1001U for 24 h. The effect of hCG treatment on the synthesis of MCF‐10F cells was monitored by labeling both control and treated cells with [S35]methionine and separation by 2‐D PAGE. At least 11 proteins were preferentially synthesized and five specific polypeptides were decreased in hCG treated cells in comparison with controls. The hCG induced at least four new mRNAs which encoded proteins in the molecular mass range of 24–72 kDa. It also increased the expression of at least six mRNAs and reduced the expression of least four mRNAs in comparision with control cells. The hCG‐treated cells actively synthesized a 33‐kDa polypeptide which was not present in control cells. The nature of this hCG‐inducible 33 kDa protein elucidated by immunoprecipating [S35]methione‐labeled proteins with antisera directed against rat inhibin subunit α and βb. The immunoprecipitation revealed a 14 kDa protein band and an 18 kDa band whose syntheses were stimulated by hCG treatment, which corresponds to the inhibin α and βb subunits, respectively.
Chiral separations by capillary electrophoresis: Present state of the art
Authors: Vespalec Radim , Boček Petr
Published: May 2005

Capillary electrophoresis is a powerful tool for chiral separation of ionogenic enantiomers in solutions. This article brings an overview of the theory of electrophoretic separations with special emphasis on enantiomer – chiral selector equilibria, followed by a survey of indirect separations, based on formation of diastereomers with different electrophoretic mobilities, and a comprehensive appraisal of direct separations when the chiral recognition stems from (i) hostguest interactions using cyclodextrins and crown ethers as hosting agents, (ii) ligand exchange mechanism, (iii) affinity interactions or (iv) a combination of solute – chiral selector interactions with micellar electrophoretic transport. Finally, some trends in chiral separations by capillary electrophoresis are discussed.
Micellar electrokinetic capillary chromatography of benzodiazepines in human urine
Authors: Schafroth Matthias , Thormann Wolfgang , Allemann Daniel
Published: May 2005

The determination of the major urinary compounds of eight common benzodiazepines, flunitrazepam, diazepam, midazolam, clonazepam, bromazepam, temazepam, oxazepam, and lorazepam, by micellar electrokinetic capillary chromatography (MECC) is shown to be a simple and attractive approach for confirmation testing of these drugs in human urine. After enzymatic hydrolysis and extraction using mixed‐mode solid‐phase cartridges and a two‐step elution protocol, fractions were analyzed in a phosphate/borate buffer (pH 9.3) containing 75 mM sodium dodecyl sulfate and small amounts of isopropanol, methanol and/or acetonitrile using an instrument with on‐ column multi‐wavelength detection. The presence of these compounds could unambiguously be confirmed in patient urines which tested positive for benzodiazepines using a commercial enzyme multiplied immunoassay screening technique (EMIT). The sensitivity of the MECC assay is demonstrated to be better than that of EMIT. MECC analysis of one patient urine which tested negatively employing EMIT revealed the presence of lorazepam, this demonstrating that false‐negative results from the initial immunological screening process can be recognized using MECC. For one example, 7‐aminoflunitra‐zepam, the MECC data are shown to agree well with those obtained by gas chromatography/mass spectrometry.
Chiral separation of basic drugs by capillary zone electrophoresis with cyclodextrin additives
Authors: Bechet Isabelle , Paques Philippe , Fillet Marianne , Hubert Philippe , Crommen Jacques
Published: May 2005

The enantiomers of a series of basic drugs were separated in capillary zone electrophoresis (CZE) in phosphate buffers, pH 3, containing β‐cyclodextrin or one of its derivatives as chiral selectors and uncoated fused silica capillaries thermostated at 15°C. The nature of the cationic component of the background electrolyte was found to have a significant influence on achiral resolution and peak symmetry. The best results were obtained with triethanolamine, which was then used to adjust the buffer pH in all further experiments. The effects on chiral resolution of the nature and concentration of cyclodextrin, of the addition of methanol, and of capillary temperature were studied. Maximum resolution was obtained at a particular cyclodextrin concentration for each analyte, depending on the affinity of the analyte for this cyclodextrin. On the basis of the results, the effects of methanol addition and temperature on enantiomeric resolution could be explained and predicted. Numerous chiral separations are presented and suggestions for the rapid optimization of CZE enantioseparations with cyclodextrin additives are given.
On the possibility of performing chiral wall‐coated open‐tubular electrochromatography in 50 μm internal diameter capillaries
Authors: Vindevogel Johan , Sandra Pat
Published: May 2005

Recent results have demonstrated acceptable enantiomer separations in open, 50 μm internal diameter capillaries, coated with a chiral stationary phase, a technique known as electrochromatography. According to existing literature, smaller column diameters, 10 μm or less, are generally preferred for this mode of operation. The conditions under which the wider columns can be used are discussed.
Screening of a mouse/human Y‐chromosomal cosmid library for gene candidates and markers by using (short) oligonucleotide probes
Authors: Träger Thorsten , Schmidt Petra , Epplen Jörg T.
Published: May 2005

A comprehensive approach is described for the identification of sequences of interest from a human Y chromosomal cosmid library via (short) consensus oligonucleotide probes. It involves the ordering of cosmid clones grown in microtiter plates onto small filter membranes by a robot workstation. A high number of the clones are characterized by their repetitive sequence content, either by ubiquitously interspersed simple tandem blocks or by Y‐specific elements. The Y chromosomal repeat (DYZ2) appears underrepresented in the library. In contrast many novel microsatellite marker systems can now be developed for the Y chromosome on the basis of the simple repeat blocks described here. Though novel genes were not yet delineated so far, a number of candidate sequences with high coding potential and other interesting characteristics are described.
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

A 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

Using 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

Glycosidases 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

A 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

We 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

We 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

Capillary 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

Preparative 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

Squamous 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

Some 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

An 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

We 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

The 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

A 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

Electrophoretic 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

On 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

The 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

The 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

This 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

Three 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.