Electrophoresis Conditions Research Articles

Study of binding constant of toll‐like receptor 4 and lipopolysaccharide using capillary zone electrophoresis

This short communication describes the interaction between toll-like receptor 4 (TLR4) and lipopolysaccharide (LPS, its specific ligand) using analytical methods. Their interaction has been evidenced in many reports. Nevertheless, there are few reports focused on their binding constant. In this research, the interaction between TLR4 and LPS is investigated using mobility shift method by CZE. To optimize the electrophoresis conditions, the effecting factors, running buffer, sample concentration, injection duration, and operation voltage of electrophoretic on the mobility shift are studied in detail. Electrophoresis conditions were described as follows: borate buffer (pH 7.4, 20 mM), 5 s for 50 mbar pressure injection duration, and 13 kV of separation voltage in 41.5 cm fused silica capillary with 75 μm id and 375 μm od. The combination constant of TLR4 and LPS is calculated using Scatchard methods. The Scatchard liner correlation is y=-0.0165x+0.1456, binding constant is K=1.65 x 10⁴ (g/mL)⁻¹.

Bis-Indole Derivatives for Polysaccharide Compositional Analysis and Chiral Resolution of D-, L-Monosaccharides by Ligand Exchange Capillary Electrophoresis Using Borate-Cyclodextrin as a Chiral Selector

A series of aldo-bis-indole derivatives (aldo-BINs) was prepared by aromatic C-alkylation reactions of aldoses and indole in acetic acid solution. Common monosaccharides such as glucose, mannose, galactose, fucose, xylose, rhamnose, ribose, arabinose and N-acetylglucosamine were smoothly derivatized to form the UV absorbing aldo-BINs. The use of a capillary electrophoretic method to separate these novel aldo-BIN derivatives was established. The capillary electrophoresis conditions were set by using borate buffer (100 mM) at high pH (pH 9.0). The limit of determination was assessed to be 25 nM. The enantioseparation of D, L-pairs of aldo-BINs based on chiral ligand-exchange capillary electrophoresis technology was also achieved by using modified hydroxypropyl-β-cyclodextrin as the chiral selector in the presence of borate buffer. This aldose labeling method was applied successfully to the compositional and configurational analysis of saccharides, exemplified by a rapid and efficient method to simultaneously analyze the composition and configuration of saccharides from the medicinal herbs Cordyceps sinensis and Dendrobium huoshanense.

Improved rapid detection of trypsin isoinhibitors using non-denaturing polyacrylamide gels with immobilised azoalbumin

Proteinaceous inhibitors of animal trypsin occur naturally as isoforms in seeds and some are of interest as antinutritional or anti-pest agents. To establish a simplified electrophorectic, in-gel method for rapid and direct detection of trypsin isoinhibitors present in crude plant extracts that are particularly suitable for many studies including rapid evaluation of cultivars. Azoalbumin (3%, w/v) is immobilised in 7.5% polyacrylamide gels before electrophoresis under non-denaturing conditions. This improved method eliminates the need for both time-consuming and labourious staining and destaining or renaturation steps. Immobilised azoalbumin in polyacrylamide gels, run under non-denaturing electrophoresis conditions, can be used to assist rapid evaluation of trypsin isoinhibitors in numerous crude plant extracts.

A d-galactose-binding lectin purified from coronate moon turban, Turbo (Lunella) coreensis, with a unique amino acid sequence and the ability to recognize lacto-series glycosphingolipids

A divalent, cation-independent d-galactose-binding lectin was purified from coronate moon turban Turbo (Lunella) coreensis. This lectin recognizes d-galactose and is a 38-kDa dimeric protein consisting disulphide-bonded 22-kDa polypeptides under non-reducing and reducing conditions of sodium dodecyl sulphate-polyacrylamide gel electrophoresis, respectively. Haemagglutination activity was inhibited by D-galactose, N-acetyl D-galactosamine, melibiose, lactose, porcine stomach mucin, asialofetuin and bovine submaxillary mucin. The lectin has tolerance for pH 5-11 and temperature until 50°C for 1h. The lectin strongly aggregated Gram-negative bacteria, such as Vibrio parahaemolyticus and Salmonella O7, but weakly Gram-positive strain as Staphylococcus aureus and Bacillus subtilis. The glycan-binding profile of this lectin was evaluated using frontal affinity chromatography technology and the lectin appeared to recognize oligosaccharides such as lacto-series glycosphingolipids contained in blood type A and H substances in addition to complex-type N-linked glycoproteins. Partial primary structures of 139 amino acid residues of this lectin were determined from N-terminus polypeptides and 8 peptides derived by cleavage with lysyl-endopeptidase. The primary structure was slightly similar to other known sequences of lectin; however, a repeating motif has been included.

Isolation, biochemical characterization, and molecular modeling of American lobster digestive cathepsin D1

An aspartic proteinase was isolated from American lobster gastric fluid. The purified cathepsin D runs as a single band on native-PAGE displaying proteolytic activity on a zymogram at pH 3.0, with an isoelectric point of 4.7. Appearance of the protein in SDS-PAGE, depended on the conditions of the gel electrophoresis. SDS treatment by itself was not able to fully unfold the protein. Thus, in SDS-PAGE the protein appeared to be heterogeneous. A few minute of boiling the sample in the presence of SDS was necessary to fully denature the protein that then run in the gel as a single band of ~50 kDa. The protein sequence of lobster cathepsin D1, as deduced from its mRNA sequence, lacks a 'polyproline loop' and β-hairpin, which are characteristic of some of its structural homologues. A comparison of amino acid sequences of digestive and non-digestive cathepsin D-like enzymes from invertebrates showed that most cathepsin D enzymes involved in food digestion, lack the polyproline loop, whereas all non-digestive cathepsin Ds, including the American lobster cathepsin D2 paralog, contain the polyproline loop. We propose that the absence or presence of this loop may be characteristic of digestive and non-digestive aspartic proteinases, respectively.

Proteomic analysis of serum of workers occupationally exposed to arsenic, cadmium, and lead for biomarker research: A preliminary study

The main factor of environmental contamination is the presence of the heavy metals lead, cadmium, and arsenic. The aim of serum protein profile analysis of people chronically exposed to heavy metals is to find protein markers of early pathological changes. The study was conducted in a group of 389 healthy men working in copper foundry and 45 age-matched non-exposed healthy men. Toxicological test samples included whole blood, serum, and urine. Thirty-seven clinical parameters were measured. Based on the parameters values of the healthy volunteers, the centroid in 37-dimensional space was calculated. The individuals in the metal-exposed and control groups were ordered based on the Euclidean distance from the centroid defined by the first component according to Principal Component Analysis (PCA). Serum samples of two individuals, one from the control and one from the metal-exposed group, were chosen for proteomic analysis. In optimized conditions of two-dimensional gel electrophoresis (2-DE), two protein maps were obtained representing both groups. Twenty-eight corresponding protein spots from both protein maps were chosen and identified based on PDQuest analysis and the SWISS-2DPAGE database. From a panel of six proteins with differences in expression greater than a factor of two, three potential markers with the highest differences were selected: hemoglobin-spot 26 (pI 7.05, Mw 10.53), unidentified protein-spot 27 (pI 6.73, Mw 10.17), and unidentified protein-spot 25 (pI 5.75, Mw 12.07). Further studies are required to prove so far obtained results. Identified proteins could serve as potential markers of preclinical changes and could be in the future included in biomonitoring of people exposed to heavy metals.

Nonaqueous capillary electrophoresis conditions for the simultaneous separation of eight alpha-adrenergic blocking agents

The analysis is described for the first time for separating eight alpha-adrenergic blocking agents (oxymetazoline, 5-methylurapidil, prazosin, phentolamine, RS-17053, methoxamine, yohimbine, and BMY7378) by capillary electrophoresis (CE) with UV detection. Optimum separation of the analytes was obtained on a 50 cm × 75 μm i.d. capillary using a buffer containing 20% acetonitrile, 60 mM ammonium acetate, and 1.0% glacial acetic in methanol medium, with applied voltage and capillary temperature of 23 kV and 25 °C, respectively. The relative standard deviations of the migration times and the peak areas of the eight analytes were in the ranges of 0.12-1.29% and 1.02-2.53%, respectively. Detection limits of oxymetazoline, 5-methylurapidil, prazosin, phentolamine, RS-17053, methoxamine, yohimbine, and BMY7378 were 0.5-1.0 μg mL(-1). In the tested concentration range, good linear relationships (correlation coefficients >98%) between peak areas and concentrations of the analytes were observed. This method has been successfully applied for determination of prazosin and phentolamine with recoveries of 97.30% and 98.12%, respectively. The proposed method was successfully applied for the rapid CE determination of the frequently applied alpha-adrenergic blocking compounds phentolamine and prazosin in general pharmaceutical preparation.

Improved DNA electrophoresis in conditions favoring polyborates and lewis acid complexation.

Spatial compression among the longer DNA fragments occurs during DNA electrophoresis in agarose and non-agarose gels when using certain ions in the conductive buffer, impairing the range of fragment sizes resolved well in a single gel. Substitutions using various polyhydroxyl anions supported the underlying phenomenon as the complexation of Lewis acids to DNA. We saw significant improvements using conditions (lithium borate 10 mM cations, pH 6.5) favoring the formation of borate polyanions and having lower conductance and Joule heating, delayed electrolyte exhaustion, faster electrophoretic run-speed, and sharper separation of DNA bands from 100bp to 12 kb in a single run.

Simultaneous determination of aminomethylbenzoic acid, cefminox sodium and etamsylate in human urine by capillary electrophoresis

A sensitive and selective capillary electrophoresis method is developed, for the first time, for effective separation and simultaneous determination of aminomethylbezoic acid (PAMBA), cefminox sodium (CMNX) and etamsylate (ETM). The electrophoresis conditions were investigated and optimized. A 25 mM phosphate solution (pH 8.5) was used as a buffer and the peak area was determined with UV detection at 216 nm wavelength under 18 kV separation voltage. Under optimal conditions, the three drugs can be separated effectively. Good linearity was achieved in 3.13–150 μg/mL for PAMBA, 6.25–150 μg/mL for CMNX and 3.13–150 μg/mL for ETM, with the correlation coefficients of >0.999. The limit of detection (LOD) for PAMBA, CMNX and ETM was 1.04, 2.08 and 1.04 μg/mL, respectively. Their recoveries in human urine were in the range from 90.2% to 101% with the RSD ( n = 5) of 0.7–3.1%. The proposed method is simple, rapid and accurate, and provides the sensitivity and linearity necessary for analysis of the test drugs in human urine at clinically relevant concentrations.

Total Trans Fatty Acid Analysis in Spreadable Cheese by Capillary Zone Electrophoresis

An alternative method for determination of total trans fatty acids expressed as elaidic acid by capillary zone electrophoresis (CZE) under indirect UV detection at 224 nm within an analysis time of 7.5 min was developed. The optimized running electrolyte includes 15.0 mmol L(-1) KH(2)PO(4)/Na(2)HPO(4) buffer (pH approximately 7.0), 4.0 mmol L(-1) SDBS, 8.0 mmol L(-1) Brij35, 45%v/v ACN, 8% methanol, and 1.5% v/v n-octanol. Baseline separation of the critical pair C18-9cis/C18:1-9t with a resolution higher than 1.5 was achieved using C15:0 as the internal standard. The optimum capillary electrophoresis (CE) conditions for the background electrolyte were established with the aid of Raman spectroscopy and experiments of a 3(2) factorial design. After response factor (R(F)) calculations, the CE method was applied to total trans fatty acid (TTFA) analysis in a hydrogenated vegetable fat (HVF) sample, and compared with the American Oil Chemists' Society (AOCS) official method by gas chromatography (GC). The methods were compared with an independent sample t test, and no significant difference was found between CE and GC methods within the 95% confidence interval for six genuine replicates of TTFA analysis (p-value > 0.05). The CE method was applied to TTFA analysis in a spreadable cheese sample. Satisfactory results were obtained, indicating that the optimized methodology can be used for trans fatty acid determination for these samples.

Separation and quantification of short-chain coenzyme A in plant tissues by capillary electrophoresis with laser-induced fluorescence detection

Coenzyme A (CoA) is a group of active metabolic compounds that facilitate over 100 chemical reactions in animal and plant cells. It mainly serves as an acyl carrier in many metabolic reactions and initiates the tricarboxylic acid cycle that produces more than 90% of the energy required for life processes. Measurements of short-chain and long-chain CoA compounds in a variety of tissues by using high-performance liquid chromatography (HPLC) and capillary electrophoresis-ultraviolet (CE-UV) detection have been reported, but these techniques do not allow one to simultaneously determine all the possible coexisting CoAs and their derivatives in plant tissues with sufficient sensitivity. In this paper, a method of quantitative determination of 5 short-chain CoAs in plant tissues by using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was developed. Under optimized derivatization and electrophoresis conditions, different CoAs that were derivatized with fluorescein-5-isothiocyanate (FITC) were separated and quantified at the pmole level. A fused silica capillary with a 75 μm (i.d.) × 57 cm was used for the separation and 150 mM borate buffer (pH 9.00) was used as a background electrolyte. The separation was carried out at 25 kV and completed in less than 13 min. The effects of derivatization time, buffer concentrations, and pH values on derivatization efficiency were also systematically investigated. This newly developed CE-LIF method can be used to detect CoAs in both plant and animal tissues.

Capillary-electrophoretic determination of zinc and cadmium ions in aqueous solutions with ion-exchange preconcentration

An approach to choosing analyte preconcentration conditions for the subsequent capillary electrophoresis (CE) analysis of the concentrate was substantiated using the simultaneous determination of zinc(II) and cadmium(II) trace concentrations as an example. A CE procedure was developed for the determination of Zn and Cd with the following characteristics: The time of the analysis, including analyte preconcentration from a 50-mL sample, was 30 min. The analytical ranges were 0.01–0.2 mg/L for cadmium(II) and 0.005–0.1 mg/L for zinc(II).

Metal–protein binding losses in proteomic studies by PAGE–LA-ICP-MS

Some experiments to study the influence of electrophoresis conditions and subsequent LA-ICP-MS (laser ablation-inductively coupled plasma mass spectrometry) determination of two metal-binding proteins with different metal-protein affinities (superoxide dismutase, containing Cu and Zn, and alcohol dehydrogenase, containing Zn) are performed. In metal-binding proteins with weak metal-protein affinities, metal losses can happen during electrophoretic separation. It has been demonstrated that the detection of these metals bound to the proteins depends, not only on the nature of the electrophoretic process (naturing or non-denaturing) and post-separation gel treatment, but also on the trailing ion chosen and current applied in the electrophoretic method used. Non-denaturing methods are preferred to denaturing ones in the case of alcohol dehydrogenase being BN-PAGE (Blue Native-Polyacrylamide Gel Electrophoresis) with the use of Tricine as trailing ion the most recommended method. The concentration obtained for Zn in ADH applying BN-PAGE-LA-ICP-MS was 2.6+/-0.30 mg g(-1) very close to the one obtained for ADH solution by ICP-MS (3+/-0.23 mg g(1)). For superoxide dismutase either denaturing or non-denaturing electrophoresis conditions can be used, but a denaturing method based on the use of Tricine as trailing ion is recommended to preserve metals-protein binding when the use of non-denaturing conditions must be avoided. The found concentration for Cu and Zn in SOD after applying SDS-Tris-Tricine-PAGE-LA-ICP-MS was 2.5+/-0.33 and 2.4+/-0.37 mg g(-1) respectively, more or less close (especially for Cu) to the one obtained in SOD solution by ICP-MS (3+/-0.21 and 3.7+/-0.32 mg g(-1) for Cu and Zn). We observe that as higher current is applied the possibility of metal-protein binding losses is higher. In all cases staining of the gel prior to LA-ICP-MS is not recommended.

Saccharomyces CDK1 Phosphorylates Rad53 Kinase in Metaphase, Influencing Cellular Morphogenesis

Rad53 is an essential protein kinase governing DNA damage and replication stress checkpoints in budding yeast. It also appears to be involved in cellular morphogenesis processes. Mass spectrometry analyses revealed that Rad53 is phosphorylated at multiple SQ/TQ and at SP/TP residues, which are typical consensus sites for phosphatidylinositol 3-kinase-related kinases and CDKs, respectively. Here we show that Clb-CDK1 phosphorylates Rad53 at Ser(774) in metaphase. This phosphorylation event does not influence the DNA damage and replication checkpoint roles of Rad53, and it is independent of the spindle assembly checkpoint network. Moreover, the Ser-to-Asp mutation, mimicking a constitutive phosphorylation state at site 774, causes sensitivity to calcofluor, supporting a functional linkage between Rad53 and cellular morphogenesis.

Repression of Ca2+/Calmodulin-dependent Protein Kinase IV Signaling Accelerates Retinoic Acid-induced Differentiation of Human Neuroblastoma Cells

Neuroblastoma cells having stem cell-like qualities are widely employed models for the study of neural stem/progenitor cell proliferation and differentiation. We find that human BE(2)C neuroblastoma cells possess a signaling cascade initiated by Ca(2+) influx via voltage-dependent calcium channels and the N-methyl-D-aspartate (NMDA) receptor and culminating in nuclear calmodulin-dependent protein kinase IV (CaMKIV)-mediated phosphorylation and activation of the transcription factors Ca(2+)/cyclic AMP-response element-binding protein (CREB) and ATF1 (activating transcription factor-1). This pathway functions to maintain BE(2)C cells in an undifferentiated, proliferative state. Parallel to this Ca(2+)-dependent pathway is a hormone-responsive program by which retinoic acid (RA) initiates the differentiation of BE(2)C cells toward a neuronal lineage. This is evidenced by RA-dependent induction of the cell cycle inhibitor p21/Cip1 (Cdk-interacting protein 1) and cell cycle arrest, induction of the neuroblastic marker doublecortin and of the neuron-specific intermediate filament protein, peripherin, and by RA-stimulated extension of neuritic processes. During neuronal differentiation there is a complex antagonistic interplay between these two major signaling pathways. RA down-regulates expression of CaMKIV and one of its upstream activators, CaMKK1 (calmodulin-dependent protein kinase kinase 1). This is accompanied by RA-induced suppression of activating phosphorylation of CREB with a time course paralleling that of CaMKIV down-regulation. RA-induced repression of the Ca(2+)/calmodulin-dependent protein kinase kinase/CaMKIV/CREB pathway appears to be involved in regulating the timing of neuronal differentiation, as shown by the effect of RNA interference of CaMKIV to markedly accelerate RA-dependent up-regulation of p21/Cip1 and doublecortin expression and RA-promoted neurite outgrowth. RA-induced repression of the CaMKIV signaling pathway may represent an early event in retinoid-dependent neuronal differentiation.

Capillary Separation: Micellar Electrokinetic Chromatography

Micellar electrokinetic chromatography (MEKC), a separation mode of capillary electrophoresis (CE), has enabled the separation of electrically neutral analytes. MEKC can be performed by adding an ionic micelle to the running solution of CE without modifying the instrument. Its separation principle is based on the differential migration of the ionic micelles and the bulk running buffer under electrophoresis conditions and on the interaction between the analyte and the micelle. Hence, MEKC's separation principle is similar to that of chromatography. MEKC is a useful technique particularly for the separation of small molecules, both neutral and charged, and yields high-efficiency separation in a short time with minimum amounts of sample and reagents. To improve the concentration sensitivity of detection, several on-line sample preconcentration techniques such as sweeping have been developed.

Calibration-free concentration determination of charged colloidal nanoparticles and determination of effective charges by capillary isotachophoresis

Although colloidal nanoparticles show an electrophoretic heterogeneity under the conditions of capillary electrophoresis, which can be either due to the particle-size distribution and/or the particle shape distribution and/or the zeta-potential distribution, they can form correct isotachophoretic zones with sharp-moving boundaries. Therefore, the technique of isotachophoresis permits to generate plugs in which the co-ions and counter ions of the original colloidal solution are removed and replaced by the buffering counter ions of the leading electrolyte. It is shown that analytical isotachophoresis can be used to measure directly, without calibration, the molar (particle) concentration of dispersed ionic colloids provided that the transference number and the mean effective charge number of the particles (within the isotachophoretic zone) can be determined with adequate accuracy. The method can also be used to measure directly the effective charge number of biomacromolecules or colloidal particles, if solutions with known molar (particle) concentration can be prepared. The validity of the approach was confirmed for a model solution containing a known molar concentration of bovine serum albumin.

Modified electrophoretic and digestion conditions allow a simplified mass spectrometric evaluation of disulfide bonds

Proper formation of disulfide bonds in proteins is a prerequisite to their stability and function. Information on disulfide pattern may therefore serve as an indication of the proper folding of recombinant proteins, and can also be used in protein homology modeling for the purpose of structure refinement. Protein handling and digestion at basic pH leads to disulfide bond scrambling. That is why the samples are usually treated and digested at low pH where no scrambling occurs. Unfortunately, the specific proteases used in protein research are active at high pH values. Here, we present a complete sample handling protocol, which allows processing of disulfide containing proteins at basic pH. We modified the standard SDS gel electrophoresis and protein digestion conditions by the addition of an oxidative agent, cystamine. This modification prevented disulfide scrambling, which we otherwise observed in the samples handled according to the general protocol. Lysozyme from hen egg was used as a model protein for the development of the method. We then applied our protocol to human leukocyte antigen CD69, for which the disulfide bonding is known, but only for its monomeric form. In addition, the disulfide arrangement was then 'de novo' identified in the recombinant murine leukocyte receptor NKR-P1A and in the larger glycosylated proteins beta-N-acetylhexosaminidases from Aspergillus oryzae and Penicillium oxalicum.

CHEF PROCEDURES: A RAPID HIGH‐TEMPERATURE METHOD FOR SAMPLE PREPARATION, A HIGH VOLTAGE HEPES BUFFER SYSTEM AND THE USE OF NUSIEVE® AGAROSE

ABSTRACT A rapid, high‐temperature method of sample preparation from gram‐negative bacteria for contour‐clamped homogenous electric field (CHEF) electrophoresis is described, which utilizes a diethylpyrocarbonate nuclease inactivation step for preventing latent degradation of DNA. Also described is a simple 4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid buffer system that permits high‐voltage CHEF electrophoresis in standard apparatus while preventing the strand scission events associated with tris(hydroxymethyl)aminomethane buffers. Finally, the use of NuSieve agarose in combination with standard analytical agarose was compared to specialty CHEF agarose, demonstrating the potential use of this reagent in CHEF gel separations. PRACTICAL APPLICATIONSContour‐clamped homogenous electric field (CHEF) gel electrophoresis is a widely used technique in epidemiological and comparative genetic studies of microorganisms. Standard techniques work variably well, making options that can be used to “tweak” methodology useful. In this article, we describe a rapid, higher‐temperature variation of sample preparation that yields inherently stable DNA with a more efficient usage of expensive reagents. Also detailed is a simple electrophoresis buffer that eliminates the tris(hydroxymethyl)aminomethane‐mediated strand scission events that have complicated the comparison of bacterial strains in some studies. This buffer permits the use of the higher‐voltage electrophoresis conditions used in many studies without the addition of potentially harmful chemicals. Finally, we demonstrate that the combination of NuSieve (Lonza) branded agarose with standard agarose can be used effectively in CHEF gels, thus expanding analytical options while also providing an alternative to maintaining other expensive specialty agaroses. The methods herein may be applied in whole or in part, allowing their possible inclusion in existing or future protocols as desired.

Optimization of capillary electrophoresis conditions for a glucagon competitive immunoassay using response surface methodology

The capillary electrophoresis (CE) conditions for a competitive immunoassay of glucagon were optimized for highest sensitivity of the immunoassay and resolution of the electrophoretic peaks using a Box-Behnken design. Injection time, voltage ramp time, and separation voltage were varied between three levels and two responses, bound-to-free (B/F) ratio of the immunoassay peaks and resolution between the peaks, were measured. Analysis of variance was applied to fit a predictive model, and a desirability function was used to simultaneously optimize both responses. A 10-s injection, 1.6-min ramp time, and a 22-kV separation voltage were the conditions found when high B/F was given more emphasis than high resolution. To test the model, calibration curves of a glucagon immunoassay were measured at the optimum and least optimum CE conditions. Optimal conditions increased the sensitivity of the immunoassay by 388% compared to the least optimum conditions while maintaining adequate resolution.