Search

Background: Tau is a microtubule associated protein that regulates the stability of microtubules and the microtubule-dependent axonal transport. Its hyperphosphorylated form is one of the hallmarks of Alzheimer's disease and other tauopathies and the major component of the paired helical filaments that form the abnormal proteinaceous tangles found in these neurodegenerative diseases. It is generally accepted that the phosphorylation extent of tau is the result of an equilibrium in the activity of protein kinases and phosphatases. Disruption of the balance between both types of enzyme activities has been assumed to be at the origin of tau hyperphosphorylation and the subsequent toxicity and progress of the disease.Objective: We explore the possibility that, beside the phosphatase action on phosphorylated tau, the catalytic subunit of PKA catalyzes both tau phosphorylation and also tau dephosphorylation, depending on the ATP/ADP ratio.Methods: We use the shift in the relative electrophoretic mobility suffered by different phosphorylated forms of tau, as a sensor of the catalytic action of the enzyme.Results: The results are in agreement with the long-known thermodynamic reversibility of the phosphorylation reaction (ATP + Protein = ADP+Phospho-Protein) catalyzed by PKA and many other protein kinases.Conclusion: The results contribute to put the compartmentalized energy state of the neuron and the mitochondrial-functions disruption upstream of tau-related pathologies. [ABSTRACT FROM AUTHOR]

The conjugation of an uncharged polymer to DNA fragments makes it possible to separate DNA by free-solution electrophoresis. This end-labeled free-solution electrophoresis method has been shown to successfully separate ssDNA with single monomer resolution up to about 110 bases. It is the aim of this paper to investigate in more detail the coupled hydrodynamic and electrophoretic deformation of the ssDNA-label conjugate at fields below 400 V/cm. Our model is an extension of the theoretical approach originally developed by Stigter and Bustamante [Biophys. J. 75, 1197 (1998)] to investigate the problems of a tethered chain stretching in a hydrodynamic flow and of the electrophoretic stretch of a tethered polyelectrolyte. These two separate models are now used together since the charged DNA is “tethered” to the uncharged polymer (and vice versa), and the resulting self-consistent model is used to predict the deformation and the electrophoretic velocity for the hybrid molecule. Our theoretical and experimental results are in good qualitative agreement. [ABSTRACT FROM AUTHOR]

We present analytic calculations of the electrophoretic mobility of stiff charged molecules in solution, and results obtained by using different approximations for the hydrodynamic interactions are discussed. For low fields, where the average molecular conformation is hardly distorted from its Gaussian shape, we find an increase of the mobility with molecular size which, for chains up to 1000 segment lengths, follows approximately a power law with an exponent B. For sufficiently high counterion concentration (λD≲segment length) B is independent of the Debye length λD. Under the simplified assumption of spherical distributed counterions we find B to be proportional to the Debye length for λD larger than the segment length, except for the limits of Gaussian and rigid rod-type molecules. A brief comparison with numerical simulations is included. © 1995 American Institute of Physics. [ABSTRACT FROM AUTHOR]

Post-translational modification with O-linked β-N-acetylglucosamine (O-GlcNAc) occurs selectively on serine and/or threonine residues of cytoplasmic and nuclear proteins, and dynamically regulates their molecular functions. Since conventional strategies to evaluate the O-GlcNAcylation level of a specific protein require time-consuming steps, the development of a rapid and easy method for the detection and quantification of an O-GlcNAcylated protein has been a challenging issue. Here, we describe a novel method in which O-GlcNAcylated and non-O-GlcNAcylated forms of proteins are separated by lectin affinity gel electrophoresis using wheat germ agglutinin (WGA), which primarily binds to N-acetylglucosamine residues. Electrophoresis of cell lysates through a gel containing copolymerized WGA selectively induced retardation of the mobility of O-GlcNAcylated proteins, thereby allowing the simultaneous visualization of both the O-GlcNAcylated and the unmodified forms of proteins. This method is therefore useful for the quantitative detection of O-GlcNAcylated proteins. [ABSTRACT FROM AUTHOR]

Ubiquitination tags proteins for different functions within the cell. One of the most abundant and studied ubiquitin modification is the Lys48 polyubiquitin chain that modifies proteins for their destruction by proteasome. In Plasmodium is proposed that post-translational regulation is fundamental for parasite development during its complex life-cycle; thus, the objective of this work was to analyze the ubiquitination during Plasmodium chabaudi intraerythrocytic stages. Ubiquitinated proteins were detected during intraerythrocytic stages of Plasmodium chabaudi by immunofluorescent microscopy, bidimensional electrophoresis (2-DE) combined with immunoblotting and mass spectrometry. All the studied stages presented protein ubiquitination and Lys48 polyubiquitination with more abundance during the schizont stage. Three ubiquitinated proteins were identified for rings, five for trophozoites and twenty for schizonts. Only proteins detected with a specific anti- Lys48 polyubiquitin antibody were selected for Mass Spectrometry analysis and two of these identified proteins were selected in order to detect the specific amino acid residues where ubiquitin is placed. Ubiquitinated proteins during the ring and trophozoite stages were related with the invasion process and in schizont proteins were related with nucleic acid metabolism, glycolysis and protein biosynthesis. Most of the ubiquitin detection was during the schizont stage and the Lys48 polyubiquitination during this stage was related to proteins that are expected to be abundant during the trophozoite stage. The evidence that these Lys48 polyubiquitinated proteins are tagged for destruction by the proteasome complex suggests that this type of post-translational modification is important in the regulation of protein abundance during the life-cycle and may also contribute to the parasite cell-cycle progression. [ABSTRACT FROM AUTHOR]

Withania somnifera is a high value medicinal plant which is used against large number of ailments. The medicinal properties of the plant attributes to a wide array of important secondary metabolites. The plant is predominantly infected with leaf spot pathogen Alternaria alternata, which leads to substantial biodeterioration of pharmaceutically important metabolites. To develop an effective strategy to combat this disease, proteomics based approach could be useful. Hence, in the present study, three different protein extraction methods tris-buffer based, phenol based and trichloroacetic acid-acetone (TCA-acetone) based method were comparatively evaluated for two-dimensional electrophoresis (2-DE) analysis of W. somnifera. TCA-acetone method was found to be most effective and was further used to identify differentially expressed proteins in response to fungal infection. Thirty-eight differentially expressed proteins were identified by matrix assisted laser desorption/ionization time of flight-mass spectrometry (MALDI TOF/TOF MS/MS). The known proteins were categorized into eight different groups based on their function and maximum proteins belonged to energy and metabolism, cell structure, stress and defense and RNA/DNA categories. Differential expression of some key proteins were also crosschecked at transcriptomic level by using qRT-PCR and were found to be consistent with the 2-DE data. These outcomes enable us to evaluate modifications that take place at the proteomic level during a compatible host pathogen interaction. The comparative proteome analysis conducted in this paper revealed the involvement of many key proteins in the process of pathogenesis and further investigation of these identified proteins could assist in the discovery of new strategies for the development of pathogen resistance in the plant. [ABSTRACT FROM AUTHOR]

The structure and state of amyloid-β peptide (Aβ) oligomers often need to be checked by reliable experimental methods. Electrophoresis is a commonly applied measurement method. However, due to the presence of detergents, oligomers are easily broken during electrophoresis, which makes it very hard to accurately assess Aβ aggregate states. In the current study, bis(sulfosuccinimidyl) suberate (BS3) was used to cross-link Aβ1–42 oligomers prior to electrophoresis. When compared to a previously reported Aβ cross-linking agent, glutaraldehyde, it was quite apparent that BS3 is more suitable for detecting intra-membrane Aβ oligomers and extra-membrane Aβ oligomers states. As such, our findings provide an efficient method for analyzing Aβ proteins or other proteins that are easily aggregated in solution and in phospholipid membranes. [ABSTRACT FROM AUTHOR]

Porphyromonas gingivalis, a periodontopathic gram-negative anaerobic bacterium, generally expresses two types of fimbriae, FimA and Mfa1. However, a novel potential fimbrilin, PGN_1808, in P. gingivalis strain ATCC 33277 was recently identified by an in silico structural homology search. In this study, we experimentally examined whether the protein formed a fimbrial structure. Anion-exchange chromatography showed that the elution peak of the protein was not identical to those of the major fimbrilins of FimA and Mfa1, indicating that PGN_1808 is not a component of these fimbriae. Electrophoretic analyses showed that PGN_1808 formed a polymer, although it was detergent and heat labile compared to FimA and Mfa1. Transmission electron microscopy showed filamentous structures (2‒3 nm × 200‒400 nm) on the cell surfaces of a PGN_1808-overexpressing P. gingivalis mutant (deficient in both FimA and Mfa1 fimbriae) and in the PGN_1808 fraction. PGN_1808 was detected in 81 of 84 wild-type strains of P. gingivalis by western blotting, suggesting that the protein is generally present in P. gingivalis. [ABSTRACT FROM AUTHOR]

Studying the effect of a magnetic field on oligonucleotide DNA can provide a novel DNA manipulation technique for potential application in bioengineering and medicine. In this work, the optical and electrochemical response of a 100 bases oligonucleotides DNA, cytosine-guanine (CG100), is investigated via exposure to different magnetic fields (250, 500, 750, and 1000 mT). As a result of the optical response of CG100 to the magnetic field, the ultra-violet-visible spectrum indicated a slight variation in the band gap of CG100 of about 0.3 eV. Raman spectroscopy showed a significant deviation in hydrogen and phosphate bonds’ vibration after exposure to the magnetic field. Oligonucleotide DNA mobility was investigated in the external electric field using the gel electrophoresis technique, which revealed a small decrease in the migration of CG100 after exposure to the magnetic field. [ABSTRACT FROM AUTHOR]

The stabilization of human papillomavirus type 16 virus-like particles has been examined by means of different techniques including dynamic and static light scattering, transmission electron microscopy and electrophoretic mobility. All these techniques provide different and often complementary perspectives about the aggregation process and generation of stabilized virus-like particles after a period of time of 48 hours at a temperature of 298 K. Interestingly, static light scattering results point towards a clear colloidal instability in the initial systems, as suggested by a negative value of the second virial coefficient. This is likely related to small repulsive electrostatic interactions among the particles, and in agreement with relatively small absolute values of the electrophoretic mobility and, hence, of the net surface charges. At this initial stage the small repulsive interactions are not able to compensate binding interactions, which tend to aggregate the particles. As time proceeds, an increase of the size of the particles is accompanied by strong increases, in absolute values, of the electrophoretic mobility and net surface charge, suggesting enhanced repulsive electrostatic interactions and, consequently, a stabilized colloidal system. These results show that electrophoretic mobility is a useful methodology that can be applied to screen the stabilization factors for virus-like particles during vaccine development. [ABSTRACT FROM AUTHOR]

In this study, we established a single nucleotide mutation matrix (SNMM) model based on the relative binding affinities of NF-κB p50 homodimer to a wild-type binding site (GGGACTTTCC) and its all single-nucleotide mutants detected with the double-stranded DNA microarray. We evaluated this model by scoring different groups of 10-bp DNA sequences with this model and analyzing the correlations between the scores and the relative binding affinities detected with three wet experiments, including the electrophoresis mobility shift assay (EMSA), the protein-binding microarray (PBM) and the systematic evolution of ligands by exponential enrichment-sequencing (SELEX-Seq). The results revealed that the SNMM scores were strongly correlated with the detected binding affinities. We also scored the DNA sequences with other three models, including the principal coordinate (PC) model, the position weight matrix scoring algorithm (PWMSA) model and the Match model, and analyzed the correlations between the scores and the detected binding affinities. In comparison with these models, the SNMM model achieved reliable results. We finally determined 0.747 as the optimal threshold for predicting the NF-κB DNA-binding sites with the SNMM model. The SNMM model thus provides a new alternative model for scoring the relative binding affinities of NF-κB to the 10-bp DNA sequences and predicting the NF-κB DNA-binding sites. [ABSTRACT FROM AUTHOR]

Small-volume continuous-flow synthesis (small-vol- Conventional Device Optimized Device nme CFS) offers a number of benefits for use in small-scale chemical production and exploratory chemistry. Typically, small-volume CFS is followed by discontinuous purification; however, a fully continuous synthesis-purification combination is more attractive. Milli free-flow electrophoresis (mFFE) is a promising continuous-flow purification technique that is well suited for integration with small-volume CFS. The purification stability of mFFE, however, needs to be significantly improved before it can be feasible for this combination. One of the major sources of instability of mFFE is attributed to the ions produced as a result of electrolysis. These ions can form pH and conductivity gradients in mFFE, which are detrimental to separation quality. The severity of these gradients has not been thoroughly studied in mFFE. In this paper, we have experimentally demonstrated that detrimental pH gradients occur at flow rates of 8 mL/ min and less, and electric field strengths of 25 V/cm and greater. To decrease the pH gradients, it is necessary to evacuate H+ and OH- as soon as they are generated; this can be done by increasing local hydrodynamic flow rates. We calculated the necessary flow rate, to be applied at the electrode, which can effectively wash away both ions before they can cause a detrimental pH gradient These optimized flow rates can be attained by designing a device that incorporates deep channels. We have confirmed the effectiveness of these channels using a prototyped device. The new design allows mFFE users to work over a wider range of flow-rate and electric-field conditions without experiencing significant changes in pH. [ABSTRACT FROM AUTHOR]

Background: Intranasal olfactory drug delivery provides a non-invasive method that bypasses the Blood-Brain-Barrier and directly delivers medication to the brain and spinal cord. However, a device designed specifically for olfactory delivery has not yet been found. Methods: In this study, a new delivery method was proposed that utilized electrophoretic forces to guide drug particles to the olfactory region. The feasibility of this method was numerically evaluated in both idealized 2-D and anatomically accurate 3-D nose models. The influence of nasal airflow, electrode strength, and drug release position were also studied on the olfactory delivery efficiency. Findings: Results showed that by applying electrophoretic forces, the dosage to the olfactory region was significantly enhanced. In both 2-D and 3-D cases, electrophoretic-guided delivery achieved olfactory dosages nearly two orders of magnitude higher than that without electrophoretic forces. Furthermore, releasing drugs into the upper half of the nostril (i.e., partial release) led to olfactory dosages two times higher than releasing drugs over the entire area of the nostril. By combining the advantages of pointed drug release and appropriate electrophoretic guidance, olfactory dosages of more than 90% were observed as compared to the extremely low olfactory dosage (<1%) with conventional inhaler devices. Conclusion: Results of this study have important implications in developing personalized olfactory delivery protocols for the treatment of neurological disorders. Moreover, a high sensitivity of olfactory dosage was observed in relation to different pointed release positions, indicating the importance of precise particle guidance for effective olfactory delivery. [ABSTRACT FROM AUTHOR]

The integration of matrix-assisted laser desorption ionization (MALDI) mass spectrometry with an upstream analytical separations (such as liquid chromatography and electrophoresis) has opened up new opportunities for the automated investigation of complex protein and peptide mixtures. The ability to efficiently analyze complex proteomic mixtures in this manner is primarily determined by the ability to preserve spatial discrimination of sample components as they leave the separation column. Current interfacing methods are problematic in this respect since minimum fraction volumes are limited to several microliters. Herein we show for the first time an LC-MALDI interface based on the formation, processing and destruction of a segmented flow. The interface consists of a droplet-generator to fractionate LC effluent into nL-volume droplets and a deposition probe that transfers the sample (and MALDI matrix) onto a conventional MALDI-MS target. The efficacy of the method is demonstrated through the analysis of Trypsin digests of both BSA and Cytochrome C, with a 50% enhancement in analytical performance when compared to conventional interface technology. [ABSTRACT FROM AUTHOR]

A series of piplartine derivatives were synthesized via Baylis-Hillman reaction and evaluated for anticancer and antibacterial activities. The cytotoxicity of these compounds was examined in two different human tumor cell lines, IMR-32 and HeLa. The antibacterial activity was examined in Staphylococcus aureus and Pseudomonas aeruginosa. The results showed that compounds 2b, 2e, and 2j were found to be the most active compounds, which displayed line no cytotoxicity, but G2-M cell cycle arrest in tumor cells, and showed cytostatic effects in bacteria. [ABSTRACT FROM AUTHOR]

Previous findings demonstrated an altered pattern of amyloid-β protein precursor (AβPP) expression in platelets of Alzheimer's disease (AD) patients compared with either healthy control subjects or patients with non-Alzheimer-type dementia. In an attempt to explore the diagnostic potential of platelet AβPP metabolism, we have generated monoclonal antibodies directed to the N-terminal part of AβPP. We have observed two different antibody recognition patterns of AβPP: one resembling previously described 130 kDa and 105 kDa species and a novel AβPP 115 kDa form. This form was significantly increased in platelets of the mild cognitive impairment and AD group as compared to control subjects. The abundance of AβPP 115 kDa species correlated with the previously described AβPP 130/105 kDa ratio as well as with Mini-Mental State Examination score. Despite the inability of these particular monoclonal antibodies to recognize native forms of AβPP, identification of a new AβPP isoform in platelets as a potential AD biomarker can provide an additional tool for the development of a reliable diagnostic test to detect preclinical stages of AD. [ABSTRACT FROM AUTHOR]

Oral Diseases (2010) , 45-52 The aim of this study was to examine whether triple depletion of salivary-α-amylase (sAA), albumin (Alb) and immunoglobulins G (IgGs) may improve the visualization capability of proteins in two-dimensional gel electrophoresis (2-DE) of oral fluids (OF). Oral fluids from healthy volunteers were subjected sequentially to sAA removing device followed by application to an Alb and IgG immunoaffinity column (triple depletion). The depleted OF samples were analyzed using SDS-PAGE followed by 2-DE and protein identification using ion-trap mass spectrometry (MS). This specific triple depletion technique unmasked spots never visualized before. A total of 36 new spots were observed after depletion (348 vs 312 before depletion). Moreover, 58 spots showed more than twofold increase intensity after depletion. In the 60-69 kDa area, the depletion procedure unmasked 14 proteins including HSP70, LTA4H, L-Plastin, Desmoplakin that are known to be involved in disease pathogenesis. The ability to selectively remove and elute the most abundant OF proteins visualized on the 2-DE represents an important step in the characterization of human OF. The better visualization and gel resolution achieved will improve quantification abilities in 2-DE and in tag-MS leading to better identification of disease-specific biomarkers. We further analyzed the eluted Alb and IgGs isoforms suggesting a new methodology venue for OF. [ABSTRACT FROM AUTHOR]

Four previously isolated di-butyl-phthalate (DBP) degraders were tested for their abilities to degrade di-(2-ethylhexyl) phthalate (DEHP). In aqueous medium supplemented with 100mg/l of DEHP, both isolate G1 and Rhodococcus rhodochrous G2 showed excellent degradative activity; in three days they were able to degrade more than 97% of the added DEHP. Rhodococcus rhodochrous G7 degraded 32.5% of the added DEHP and Corynebacterium nitrilophilus G11 showed the least amount of DEHP degradation. The addition of surfactant Brij 30 at 0.1× critical micelle concentration (2mg/l) significantly improved DEHP degradation by Rhodococcus rhodochrous G2 (more than 90% of the added DEHP was degraded within 24 hours), but slightly inhibited the degradation of DEHP by the isolate G1 and Rhodococcus rhodochrous G7. Based on the 16S rDNA sequence data, isolate G1 was identified as Gordonia polyisoprenivorans. Soil inhibited DEHP degradation by G. polyisoprenivorans G1; fourteen days after a second addition of DEHP, 11.5% of the total added DEHP (i.e., 243.4μg/g soil) remained detectable. Changes in the bacterial community were monitored using denaturing gradient gel electrophoresis (DGGE) and respective dendrogram analysis. It is clear that DEHP and DEHP plus G. polyisoprenivorans G1 substantially affected the bacterial community structure in the soils. However, as the population of indigenous DEHP degraders increased in the DEPH-treated soil, its bacterial communities resembled those in the DEHP plus G. polyisoprenivorans G1-inoculated soil by Day 17. [Copyright &y& Elsevier]

This paper shows two types of microcapsules used for electrophoretic display. One is prepared by in-situ polymerization which is based on urea, melamine and formaldehyde and another by complex coacervation, which is composed of gelatin and gum Arabic. Microcapsules attract interests of many research groups for longer lifetime of electrophoretic display by reducing agglomerization or lateral movements of nanoparticles. The gelatin microcapsules were more attractive in providing more uniform microcapsule coverage on electrodes due to their flexibility as compared to the melamine-urea microcapsules. The properties of microcapsules were characterized by FTIR, OM, SEM and TGA. Migration of nanoparticles in the two types of microcapsules was also observed when an electric field was applied. [ABSTRACT FROM AUTHOR]

A novel method for performing electrophoretic separations is described-gradient elution moving boundary electrophoresis (GEMBE). The technique utilizes the electrophoretic migration of chemical species in combination with variable hydrodynamic bulk counterflow of the solution through a separation capillary or microfluidic channel. Continuous sample introduction is used, eliminating the need for a sample injection mechanism. Only analytes with an electrophoretic velocity greater than the counterflow velocity enter the separation channel. The counterfiow velocity is varied over time so that each analyte is brought into the separation column at different times, allowing for high-resolution separations in very short channels. The new variable of bulk flow acceleration affords a new selectivity parameter to electrophoresis analogous to gradient elution compositions in chromatography. Because it does not require extra channels or access ports to form an injection zone and because separations can be performed in very short channels, GEMBE separations can be implemented in much smaller areas on a microfluidic chip as compared to conventional capillary electrophoresis. Demonstrations of GEMBE separations of small dye molecules, amino acids, DNA, and immunoassay products are presented. A low-cost, polymeric, eight-channel multiplexed microfluidic device was fabricated to demonstrate the reduced area requirements of GEMBE; the device was less than 1 in. in area and required only n + 1 fluidic access ports per n analyses (in this instance, nine ports for eight analyses). Parallel separations of fluorescein and carboxyfluorescein yielded less than 3% relative standard deviation (RSD) in interchannel migration times and less than 5% RSD in both peak and height measurements. The device was also used to generate a calibration curve for a homogeneous insulin immunoassay using each of the eight channels as a calibration point with less than 5% RSD at each point with replicate analyses. [ABSTRACT FROM AUTHOR]

The safe and efficient delivery of DNA remains the major barrier to the clinical application of non-viral gene therapy. Here, we present novel, biodegradable polymers for gene delivery that are capable of simple graft modification and demonstrate the ability to respond to intracellular conditions. We synthesized poly(β-amino ester)s using a new amine monomer, 2-(pyridyldithio)-ethylamine (PDA). These cationic, degradable polymers contain pyridyldithio functionalities in the side chains that react with high specificity toward thiol ligands. This reactivity is demonstrated using both mercaptoethylamine (MEA) and the thiol peptide RGDC, a ligand that binds with high affinity to certain integrin receptors. These two polymer derivatives displayed strong DNA binding as determined using electrophoresis and dye exclusion assays. In addition, the MEA-based polymer and plasmid DNA were shown to self-assemble into cationic complexes with effective diameters as low as 100 nm. Furthermore, this DNA binding ability was substantially reduced in response to intracellular glutathione concentrations, which may aid in DNA unpackaging inside the cell. These complexes also displayed low cellular toxicity and were able to mediate transfection at levels comparable to PEI in human hepatocellular carcinoma cells. These results suggest that PDA-based poly-(β-amino ester)s may serve as a modular platform for polymer-mediated gene delivery. [ABSTRACT FROM AUTHOR]

This paper demonstrates the ability to use capillary electrophoresis (CE) separation coupled with laser-induced fluorescence for analyzing the contents of single femtoliter-volume aqueous droplets. A single droplet was formed using a T-channel (3 μm wide by 3 μm tall) connected to microinjectors, and then the droplet was fluidically moved to an immiscible boundary that isolates the CE channel (50 μm wide by 50 μm tall) from the droplet generation region. Fusion of the aqueous droplet with the immiscible boundary effectively injects the droplet content into the separation channel. In addition to injecting the contents of droplets, we found aqueous samples can be introduced directly into the separation channel by reversibly penetrating. and resealing the immiscible partition. Because droplet generation in channels requires hydrophobic surfaces, we have also investigated the advantages to using all hydrophobic channels versus channel systems with patterned hydrophobic and hydrophilic regions. To fabricate devices with patterned surface chemistry, we have developed a simple strategy based on differential wetting to deposit selectively a hydrophilic polymer (poly(styrenesulfonate)) onto desired regions of the microfluidic chip. Finally, we applied our device to the separation of a simple mixture of fluorescein-labeled amino acids contained within a ∼-10-fL droplet. [ABSTRACT FROM AUTHOR]

A novel method for highly sensitive detection of primary and secondary amino acids with selective derivatization using acetaldehyde as a new derivatization reagent was proposed by capillary electrophoresis (CE) coupled with electrogenerated chemiluminescence (ECL) of tris(2,2′-bipyridine)ruthenium(II). The precolumn derivatization of these amino acids with acetaldehyde was performed in aqueous solution at room temperature for 1 h. Upon optimized derivatization, the ECL intensities and detection sensitivities of the amino acids were significantly enhanced by 20–70 times. Using four amino acids, arginine, proline, valine, and leucine, as model compounds, their derivatives could be completely separated by CE and sensitively detected by ECL within 22 min. The linear ranges were 0.5–100 μM for arginine and proline and 5–1000 μM for valine and leucine with the detection limits of 1 × 10-7 (0.5 fmol, arginine), 8 × 10-8 (0.4 fmol, proline), 1 × 10-6 (5 fmol, valine), and 1.6 × 10-6 M (8 fmol, leucine) at a signal-to-noise ratio of 3. The derivatization reactions and ECL process of amino acids were also proposed based on in situ Fourier transform infrared and ultraviolet spectrometric analyses. [ABSTRACT FROM AUTHOR]

While temperature control is usually employed in capillary electrophoresis (CE) to aid heat dissipation and provide acceptable precision, internal electrolyte temperatures are almost never measured. In principle, this limits the accuracy, repeatability, and method robustness. This work presents a fundamental study that combines the development of new equations characterizing temperature profiles in CE with a new method of temperature determination. New equations were derived from first principles relating the mean, axial, and inner wall electrolyte temperatures (TMean, TAxis, TWall). TMean was shown to occur at a distance 1/√3 times the internal radius of the capillary from the center of the capillary and to be a weighted average of 2⁄3TAxis and 1⁄3TWall. Conductance (G) and electroosmotic mobility (PEOF) Can be used to determine TMean and TWall, respectively. Extrapolation of curves of μEOF versus power per unit length (P/L) at different temperatures was used to calibrate the variation of μEOF with temperature (T), free from Joule heating effects. μEOF increased at 2.22%/°C. The experimentally determined temperatures using μEOF agreed to within 0.2 °C with those determined using G. The accuracy of G measurements was confirmed independently by measuring the electrical conductivity (κ) of the bulk electrolyte over a range of temperatures and by calculating the variation of G with T from the Debye-Hückel-Onsager equation. TMean was found to be up to 20 °C higher than the external temperature under typical conditions using active air-cooling and a 74.0-μm-internal diameter (di) fused-silica capillary. A combination of experimentally determined and calculated temperatures enables a complete temperature profile for a fused-silica capillary to be drawn and the thickness of the stationary air layer to be determined. As an example, at P/L = 1.00 Wm-1, the determined radial temperature difference across the electrolyte was 0.14 °C; the temperature difference across the fused-silica wall was 0.17 °C, across the poly(imide) coating was 0.13 °C, and across the stationary air layer was 2.33 °C. [ABSTRACT FROM AUTHOR]

A novel strategy for screening the enzyme inhibitors from the complex mixtures by capillary electrophoresis with an on-column immobilized enzyme microreactor created by an ionic binding technique is reported. The enzyme microreactor was prepared in two steps: First, the capillary wall was dynamically coated with a polycationic electrolyte hexadimethrine bromide (HDB) by simply flushing the column using the HDB solution. Subsequently, a plug of the enzyme solution was injected and incubated for 5 min to permit the enzyme molecules to immobilize on the positively charged coating via ionic binding. To demonstrate this strategy, angiotensin-converting enzyme (ACE) was employed as a model for the enzyme immobilization, inhibition study, and inhibitor screening. It has been proved that such a prepared immobilized ACE microreactor displays a high enough activity and stability. Furthermore, the immobilized enzyme microreactor could be easily renewed. The inhibition study or inhibitor screening was accomplished through the following procedure: (i) the substrate solution was injected and incubated within the microreactor for a short time span; (ii) subsequently, the voltage was applied to separate the product of the enzyme reaction from the unreacted substrate based on their different mobilities, the peak area of the product representing the enzyme activity; (iii) a certain amount of enzyme inhibitor or candidate compound was spiked into the substrate solution to assay the reduction of the immobilized enzyme activity. Thus, the inhibitors can be easily identified if the reduced peak area of the product is observed in electropherograms. Because the injection volume of the capillary was only 9.8 nL and the enzyme could be reusable, the assay cost could be dramatically reduced. The screening of a small compound library containing natural extracts and commercially available inhibitors was performed. The present approach has proved to be simple, rapid, and robust. [ABSTRACT FROM AUTHOR]

Electrophoretic separation of a mixture of chemical species is a fundamental technique of great usefulness in biology, health care, and forensics. In capillary electrophoresis (which has evolved from its predecessor, slab-gel electrophoresis), the sample migrates through a single microcapillary instead of through the network of pores in a gel. A fundamental design problem is to minimize dispersion in the separation direction. Molecular diffusion is inevitable and sets a theoretical limit on the best separation that can be achieved. But in practice, there are a number of effects arising out of the interplay between fluid flow, chemistry, thermal effects, and electric fields that result in enhanced dispersion. This paper reviews the subject of fluid flow in such capillary microchannels and examines the various causes of enhanced dispersion that limit the efficiency of separation. [ABSTRACT FROM AUTHOR]

Animal glues were often used in historic and artistic objects, e.g. as paint ground, as binders for pigments, or as adhesives. The sources were egg, casein, or different collagens. For restoration and conservation purposes it is important to know which kind of animal glue a museum object contains. Capillary electrophoresis can deliver such information, because it enables differentiation among the three proteinaceous glue classes according to their different amino acid patterns after hydrolysis. This work deals with the most relevant problem in practice, whether this identification is obstructed by the presence of other binders, with which they are mixed in many real samples; in particular, interference from plant gums and drying oils was investigated. Capillary electrophoresis of the hydrolysates (after reaction with 6 mol L−1 HCl) was performed with an acidic background electrolyte consisting of chloroacetic acid (51.9 mmol L−1) adjusted with LiOH to pH 2.26. The underivatised analytes were detected with a contactless conductivity detector. It was found that the constituents of the plant gums (monosaccharides) or drying oils (long-chain fatty acids and short-chain dicarboxylic acids) never interfered with identification of the animal glues, as shown for artificial mixtures of the different binders even at tenfold excess over the animal glue, and for egg tempera samples. The method was used to identify the filling material from a statue from the eighteenth century. [ABSTRACT FROM AUTHOR]

A simple and sensitive method is described for determination of jasmonic acid (JA) in plant tissues. The method is based on derivatization of JA with 5-bromomethylfluorescein (5-BMF) and separation and quantification of the resulting 5-BMF–JA derivative by capillary electrophoresis coupled to laser-induced fluorescence detection (CE–LIF). The derivatization conditions were studied in detail. Our results indicated that 5-BMF-labeled JA could be well separated from other plant hormones present in the sample by use of 20 mmol L−1 borate buffer (pH 8.5). The response to JA was a linear function of concentration in the range 1 to 100 μmol L−1, with a correlation of 0.9986. Our preliminary work showed that the proposed method had fairly good selectivity and sensitivity. Only small amounts of plant sample are needed to complete the analysis. This described method enables the analysis of JA in crude extracts without extra purification and enrichment procedures. [ABSTRACT FROM AUTHOR]

A fast capillary zone electrophoresis (CZE) method was developed for the determination and separation of saikosaponins a, c, and d in Chinese herbal extracts of Bupleurum Chinese DC from different areas. Detection at 214 nm with a system containing sodium borate buffer and mono-3-phenylcarbamoyl-β-CD was found to be the most suitable approach for this analysis. Saikosaponins a, c, and d could be easily determined within 8 min. The effect of the concentration of mono-3-phenylcarbamoyl-β-CD, the concentration of the running buffer and buffer pH value on the migration behavior of the saikosaponins is discussed. [ABSTRACT FROM AUTHOR]

State-of-the-art microfluidic analytical systems are briefly surveyed. Attention is focused on the use of microchip capillary electrophoresis. The main results obtained in the development of a prototype analytical system with a laser-induced fluorescence detector for electrophoresis on a glass microfluidic chip are presented. Experimental data on electroosmotic flow and the distribution of sample fluorescence intensity over the cross section of a microchannel are analyzed. A procedure for the rapid analysis of oligonucleotides on a microfluidic chip is described. [ABSTRACT FROM AUTHOR]

Following the pioneering use of lasers and photon beating for the development of dynamic light scattering about thirty years ago, there have been numerous studies using light scattering techniques to investigate liquid dispersions in various research fields. With the growing importance of new polymeric and colloidal materials in technology and biopolymers and biocolloids in biology, the use of these techniques for size characterization has become routine. This paper gives a survey on basic principles of light scattering techniques and their use in physics, chemistry, biochemistry, and pharmaceutical research. We will emphasize recent applications and the combination of light scattering with other techniques such as electrophoresis, chromatography, and flow cytometry. Finally, although light scattering shows several advantages compared to other techniques for characterization of nanoparticles, critical observations are discussed with respect to its potentials and pitfalls. [ABSTRACT FROM AUTHOR]

Flow Analysis was started in 1979, first held in Amsterdam. A brief history of the conference is presented, along with the important roles flow analysis plays in analytical measurements. Flow analysis comprises numerous techniques that encompass much of what we do as analytical chemists. It includes operations and techniques such as segmented, continuous or stopped flow, chromatography, and electrophoresis. Flow methods are used for handling and treating solutions, and for separations, and are applicable for all sorts of wet chemical operations and measurements. They provide rapid, precise, and automated measurements, using small samples, with low reagent consumption. They form the basis of total microanalysis systems. [Copyright &y& Elsevier]

The formation and transformation processes of isopolyoxotungstates have been studied in aqueous and aqueous-CH3CN solutions of 5.0 mM W(VI), using capillary electrophoresis (CE). In freshly prepared aqueous media of pH 2.0–5.0, five migration peaks were identified as [W10O32]4−, ψ-metatungstate, β-[H2W12O40]6−, α-[H2W12O40]6− and [W7O24]6−. In the pH region of 2.0–4.0, the time-dependence of each peak area corresponds to the spontaneous direction of transformation: [W10O32]4−→ψ-metatungstate→β-[H2W12O40]6−→α-[H2W12O40]6−. At higher pH values, [W7O24]6− also showed the same sequence of transformation: [W7O24]6−→ψ-metatungstate→β-[H2W12O40]6−→α-[H2W12O40]6−. In 40% (v/v) CH3CN–water media, [W10O32]4− and [W6O19]2− were kinetically stable in the buffer pH range of 2.0–3.5. By heating the aqueous-CH3CN solutions at buffer pH values of 4.0–5.0, a mixture of α-[H4W12O40]4− and [W7O24]6− was formed. On the other hand, ψ-metatungstate, β-[H2W12O40]6− and α-[H2W12O40]6− did not exist at any appreciable concentrations. Their formation conditions were established as functions of the buffer pH, the reaction time, the reaction temperature and the CH3CN concentration. [Copyright &y& Elsevier]

A critical discussion about the possibility of improving the method validation process by means of experimental design is presented. The reported multivariate strategies concern the evaluation of the performance parameters robustness and intermediate precision, and the optimisation of bias and repeatability. In particular, accuracy and precision improvement constitutes a special subset of experimental design in which the bias and the relative standard deviation of the assay are optimised. D-optimal design was used in order to plan experiments for this aim. The analytical methods considered were capillary electrophoresis, HPLC, adsorptive stripping voltammetry and differential pulse polarography. All methods were applied to real pharmaceutical analysis problems. [ABSTRACT FROM AUTHOR]

Describes the chemical applications of a programmable image acquisition system. Identification of compounds in commercial drugs; Analysis of the electrophoresis bands; Amplification of a particular gene using PCR techniques.

The aim of this work was to determine inorganic ions from natural waters by capillary electrophoresis (CE) and to compare the results obtained with those measured with conventional solvent chemistry techniques. The project was part of a larger CE study, during which we measured inorganic ions from some lake and river systems and groundwaters in Southern Finland. Results obtained from contaminated Finnish waters were compared with samples from the River Rhine in the Du¨sseldorf area. Two CE methods were used for analysis: one for determination of chloride, sulfate, nitrite and nitrate at pH 7.7 and the other for ammonium, potassium, calcium, sodium and magnesium at pH 3.6, both methods using identification based on indirect UV detection. Two separation methods were used in order to prevent complex formation of metals with sulfate, hydroxide and decomposed organic matter present in the environmental samples. On the basis of the CE studies dilution was needed for those samples having more than 100 mg/l of sulfate, chloride, calcium and sodium. On average, the natural waters in the study contained ammonium, magnesium, sodium, potassium and calcium below 0.3, 20, 200, 20, and 200 mg/l, respectively. The concentrations of chloride, sulfate, nitrite and nitrate were below 20, 100, 10, and 10 mg/l, respectively. Correlation of the CE results with those acquired by titration, atomic absorption spectrometry, ion chromatography and flow injection analysis were obtained; R2 values for the comparison tests varied from 0.8816 to 0.9994 depending on the ion. The repeatabilities of the anion and cation CE methods were tested using laboratory-made reference sample mixtures with high and low salt concentration. [Copyright &y& Elsevier]

Features abstracts of papers on chemistry presented at the 2000 Pittsburgh Conference, published in the 2001 issue of the periodical 'Journal of Automated Methods & Management in Chemistry.' Use of x-ray lithography and injection moulding to transfer the design of the microfabricated device to a plating base; Development of an optically gated electrophoresis on a chip.

Generating an expected longitudinal pH distribution is of essential importance for achieving high resolution in an eiectrophoresis process conducted in a multicompartment eiectrolyzer. A simulation of the longitudinal distribution of pH in a multicompartment eiectrolyzer is proposed on the basis of mass transfer analysis and electrolysis equilibrium of the electrolytes. Selection of the operating conditions to establish an expected pH distribution with a conventional electrolyte rather than an ampholyte can thus be accomplished according to the simulation results. Numerical solution of this model is developed on the basis of the Gear method. The calculated results agree well with the experimental data in a wide range of operation conditions including applied potential, sample loading flow rate, and protein concentration in solution. This model may also be applied to other ampholyte-free eiectrophoresis processes conducted in a membrane-spaced multicompartment eiectrolyzer. [ABSTRACT FROM AUTHOR]

1. Proteins of fat-globule membrane from bovine milk were solubilized with the non-ionic detergent Triton X-100 in the presence of protease inhibitors. Approximately 25% of the total membrane protein was solubilized and the extracts were shown to contain a sample of most of the major membrane proteins and glycoproteins. 2. The solubilized proteins were separated in flat-beds of Ultrodex by electrofocusing and the pI values for the major proteins, glycoproteins and certain enzymes determined. Several of the proteins displayed marked heterogeneity indicating the existence of protein variants and isoenzymes. Principal pl values for the enzymes assayed were as follows: xanthine oxidase, 7.35–7.55; NADH2:iodonitrotetrazolium reductase, less than 4.5; 5′-nucleotidase, 7.15–7.4; alkaline phosphatase, 5.4–5.7; phosphodiesterase, 4.6–4.8; γ-glutamyl transpeptidase, 4.4–4.55. 3. Fractions after electrofocusing were analyzed by ‘fused rocket’ immunoelectrophoresis and crossed immunoelectrophoresis after separation in polyacrylamide gels containing sodium dodecyl sulphate. Major antigens of the membrane include xanthine oxidase and glycoproteins of apparent molecular weights 67000, 49500 and 46000. The latter two components share common antigenic determinants and could not be separated by gel filtration, ion-exchange chromatography, lectin-affinity chromatography or preparative electrofocusing. [ABSTRACT FROM AUTHOR]

Twenty-three proteins were identified by two-dimensional electrophoresis on polyacrylamidegel slabs in the 30-S ribosomal subunit of Bacillus stearothermophilus strain 799. They were designated as B-S1 through B-S21, B-Sa and B-Sb and purified on carboxymethyl-cellulose and Sephadex G100 in the presence of 6 M urea. Their molecular weight was estimated by dodecylsulfate-gel electrophoresis and their amino acid composition was determined after acid hydrolysis. Results obtained for the individual proteins were essentially similar to those for Escherichia coli 30-S proteins with some characteristic differences. [ABSTRACT FROM AUTHOR]

We studied the linearity and detection limits of the capillary zone electrophoresis system Capillarys® in the measurement of serum monoclonal protein. Three monoclonal proteins with different isotypes and electrophoretic migrations were diluted with a hypo-γ-globulinemic polyclonal serum pool. Mathematical linearity was observed for all monoclonal protein isotypes in the ranges studied without removing the polyclonal γ-globulin background. Theoretical concentrations of 0.43, 0.89 and 0.33 g/L for monoclonal proteins immunoglobulin (Ig)G, IgA and IgM, respectively, gave a discernible spike by Capillarys®, although they were measured as 0.76, 1.09 and 0.76g/L, respectively. We observed overestimation of monoclonal protein inversely correlated to concentrations below 15 g/L. All these limitations have to be taken into account when monitoring monoclonal proteins, because the loss of linearity and the protein background may hide an increase in concentration at low levels. [ABSTRACT FROM AUTHOR]

Iodohalides and halides of the choline series were separated by capillary electrophoresis using an unmodified fused-silica capillary. The developed procedures ensure the determination of iodohalides in the presence of ammonium ions. They were tested in the analysis of model pharmaceuticals of the composition organic iodohalide : polyvinyl pyrrolidone (PVP) (1 : 4). The proposed procedure ensures the estimation of the concentration of choline-mimetic substances in the range from 1 × 10–5 to 5 × 10–3 M. [ABSTRACT FROM AUTHOR]