Cesium reversibly suppresses HeLa cell proliferation by inhibiting cellular metabolism.

The aim of the present study was to investigate the influence of Cs on cultured human cells. We find that HeLa cell growth is suppressed by the addition of 10 mm CsCl into the culture media. In the Cs -treated cells, the intracellular Cs and K concentrations are increased and decreased, respectively.

Capillary isoelectric focusing after sample enrichment with immunoaffinity chromatography in a single capillary.

For accurate micro-scale quantification of a specific protein in biological fluids, immunoaffinity chromatography (IAC) and isoelectric focusing (IEF) were combined in a single fused-silica capillary. The inner wall of the capillary was coated with an anti-E-tag antibody at the inlet side to form an IAC column, and polydimethylacrylamide, a neutral polymer, at the outlet side to form the capillary for IEF. After loading a sample, the whole capillary was filled with a carrier ampholyte solution.

Affinity probe capillary electrophoresis of insulin using a fluorescence-labeled recombinant Fab as an affinity probe.

Affinity probe CE (APCE) separates and detects a target molecule as a complex using a fluorescence-labeled affinity probe (AP) by CE. The electrophoretic separation of the complex ensures accurate identification of a specific signal among nonspecific ones, which often compromises the credibility of immunoassays. APCE of insulin using a recombinant Fab (rFab) as an AP was demonstrated as a model system in this report.

Estimation of the deamidation rates of major deamidation sites in a Fab fragment of mouse IgG1-κ by capillary isoelectric focusing of mutated Fab fragments.

The deamidation of asparagine (Asn or N) residues in proteins is a common post-translational chemical modification. The identification of deamidation sites and determination of the degree of deamidation have been carried out by the combination of peptide mapping and mass spectrometry. However, when a peptide fragment contains multiple amides, such analysis becomes difficult and sometimes impossible.

Charge heterogeneity of a therapeutic monoclonal antibody conjugated with a cytotoxic antitumor antibiotic, calicheamicin.

A robust and highly reproducible capillary isoelectric focusing (cIEF) method for the evaluation of charge heterogeneity of monoclonal antibody (mAb) pharmaceutical which contains covalently bound antitumor compounds was developed using a combination of commercially available dimethylpolysiloxane-coated capillary and carrier ampholyte. In order to optimize major analytical parameters for robust mobilization, experimental responses from three pI markers were selected. The optimized method gave excellent repeatability and intermediate precision in estimated pI values of charge variants with relative standard deviations (RSDs) of not more than 0.

Two-step perpendicular free-solution isoelectric focusing in a microchamber array chip.

A free-solution microfluidic chip device was fabricated for small-scale protein fractionation based on the principle of two-step perpendicular isoelectric focusing (IEF) previously reported in a gel slab. The microchip was composed of two separate glass plates with a square array of 100 indentations etched on the facing side of each plate. By changing the relative position of the two plates, ten channels can be formed as ranges of the staggered indentations and can be switched to the perpendicular direction, or 100 chambers of 140 nL each can be produced by perfect overlapping of the indentations.

Micro OS-ELISA: Rapid noncompetitive detection of a small biomarker peptide by open-sandwich enzyme-linked immunosorbent assay (OS-ELISA) integrated into microfluidic device.

A novel detection system that combines the merits of open-sandwich (OS) enzyme-linked immunoadsorbent assay (ELISA) and a microfluidic sensor chip system, and which enables rapid and noncompetitive immunodetection of small antigens of less than 1000 in molecular weight, has been proposed. Equipped with a sensitive thermal lens microscope, a minute amount of the carboxyl-terminal peptide of human osteocalcin (BGP), a biomarker for bone metabolism, was quantified utilizing antigen-dependent stabilization of an antibody variable region (OS principle). In a short analysis time (approximately 12 min), we could attain a detection limit comparable to that of the microplate-based OS ELISA (1 microg L(-1)).

A capillary holder for scanning detection of capillary isoelectric focusing with laser-induced fluorescence.

A holder for a 12 cm long capillary was designed for scanning LIF detection of CIEF. The polyimide coat of a fused-silica capillary has been removed, and 1.5 mm diameter flanges have been attached near both ends.

Recent advances in IEF in capillary tubes and microchips.

The methodological advancements in CIEF in tubes and microchips during the period between 2002 and early 2008 are reviewed as a continuation of previous review by the same author (Electrophoresis 2002, 23, 3847-3857). After a brief introduction, the following topics related to CIEF technologies are addressed: the materials used to form capillary tubes and chips; modes of CIEF related to the detection schemes; coatings of the capillary walls; additives to the separation media; sample-loading methods; development of pI markers and their use; focusing time in relation to the length of the pH gradient; resolution in terms of a peak capacity; and, the reproducibility and precision of CIEF. Three principal detection methods are examined, i.

Isoelectric focusing in a microfluidically defined electrophoresis channel.

A new form of microchip isoelectric focusing that allows efficient coupling with pretreatment processes is reported. The sample is conveyed in a carrier ampholyte solution to the separation channel that is connected at both ends by two V-shaped lead channels, which supply electrode solutions to the connection point and complete the electrical connection to off-chip electrodes. The relatively high electric conductivity of the electrode solutions compared with that of the pH gradient enables focusing with a 2% loss of applied voltage at the electrodes using the lead channels.

Pesticide analysis by MEKC on a microchip with hydrodynamic injection from organic extract.

An integrated microchip for monitoring carbamate pesticides in environmental water using continuous flow chemical processes is under development, i. e., the integration of hydrolysis, azo-derivatization, liquid-liquid extraction, electrophoretic separation, and quantification.

On-chip connector valve for immunoaffinity chromatography in a microfluidic chip.

A miniature valve that operates between a chip port and a tube fitting was developed. The valve functions by means of a rotor, 3 mm in diameter and 1.5 mm in height, made of Teflon, with a 0.

Application of a micro multiphase laminar flow on a microchip for extraction and determination of derivatized carbamate pesticides.

Determination of carbamate pesticides such as carbaryl, carbofuran, propoxur and bendiocarb was demonstrated on a microchip with newly designed microchannels developed for efficient solvent extraction. The pesticides were hydrolyzed to corresponding naphthols, coupled with p-nitrobenzenediazonium fluoroborate reagent, and then extracted into 1-butanol as colored azo derivatives and detected with a thermal lens microscope. Optimum flow rates for the aqueous and organic phases were evaluated in the continuous-flow chemical process established in the microchip.

Analysis of protein-protein interactions with a multi-capillary electrophoresis instrument.

Protein-protein interactions were analyzed by zone electrophoresis of premixed equilibrium mixtures of a fluorescence-labeled protein at a constant concentration and unlabeled protein at a variety of concentrations using a 96-CE instrument equipped with a LIF detector. The interactions between labeled-con A versus succinylated ovalbumin, labeled-trypsin versus four proteinaceous trypsin inhibitors and labeled-insulin versus seven anti-insulin monoclonal antibodies were analyzed using a dual buffer system, in which a 60 mM borate-Na buffer (pH 9.35) was used as electrophoresis buffer and 60 mM MOPS-Na (pH 7.

Mobility moment analysis of molecular interactions by capillary electrophoresis.

A new method for the quantitative evaluation of molecular interactions that are observed in electrophoresis is described. One component taking part in the interaction is labeled with a fluorescent dye and is subjected to capillary zone electrophoresis with fluorescence detection in the presence or absence of an unlabeled interacting component. Fluorescence signals are collected at constant time intervals, and the electropherograms are converted to represent the fluorescence signal against mobility.

Recent advances in capillary isoelectric focusing: 1997-2001.

The methodological developments in the field of capillary isoelectric focusing (CIEF) published between 1997-2001 are reviewed as a continuation of the previous review by Rodriguez-Diaz et al. (Electrophoresis 1997, 18, 2134-2144). The applications are summarized and the progress in CIEF technologies, including experimental setup with coated and uncoated capillaries, remedies for the presence of salts in samples, additives to reduce precipitation of samples during the focusing process, calibration of the pH gradients, issues of reproducibility, carrier ampholyte-free CIEF, and a computer simulation of focusing process are discussed.

Determination of the affinity constants of recombinant human galectin-1 and -3 for simple saccharides by capillary affinophoresis.

The affinity constants of recombinant human galectin-1 and galectin-3 for sugars were determined by capillary affinophoresis. The monoliganded affinophore contains p-aminophenyl-beta-lactoside as an affinity ligand in the matrix of succinylglutathione and has three negative charges. An analysis of the mobility change of the lectins caused by the affinophore and its inhibition by neutral sugars allowed, for the first time, a determination of the affinity constants between the binding sites of the lectins and sugars.

Fluorescence-labeled peptide pI markers for capillary isoelectric focusing.

Nineteen fluorescent pH standards or pI markers ranging pH 3.64-10.12 were developed for use in capillary isoelectric focusing using laser-induced fluorescence detection.

Immunoassay of serum alpha(1)-antitrypsin by affinity-probe capillary isoelectric focusing using a fluorescence-labeled recombinant antibody fragment.

An immunoassay for human alpha(1)-antitrypsin based on affinity-probe capillary isoelectric focusing (AP-CIEF) is described. The method is based on the separation of free and bound labeled antibody fragments by CIEF with laser-induced fluorescence detection. A recombinant Fab' fragment of mouse immunoglobulin G1 (IgG1) against human alpha(1)-antitrypsin was labeled with tetramethylrhodamine on the single cysteine residue at the hinge region.