A rapid and efficient two-step gel electrophoresis method for the purification of major rye grass pollen allergens

Purified proteins are mandatory for molecular, immunological and cellular studies. However, purification of proteins from complex mixtures requires specialised chromatography methods (i.e., gel filtration, ion exchange, etc.) using fast protein liquid chromatography (FPLC) or high-performance liquid chromatography (HPLC) systems. Such systems are expensive and certain proteins require two or more different steps for sufficient purity and generally result in low recovery. The aim of this study was to develop a rapid, inexpensive and efficient gel-electrophoresis-based protein purification method using basic and readily available laboratory equipment. We have used crude rye grass pollen extract to purify the major allergens Lol p 1 and Lol p 5 as the model protein candidates. Total proteins were resolved on large primary gel and Coomassie Brilliant Blue (CBB)-stained Lol p 1/5 allergens were excised and purified on a secondary "mini"-gel. Purified proteins were extracted from unstained separating gels and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analyses. Silver-stained SDS-PAGE gels resolved pure proteins (i.e., 875 μg of Lol p 1 recovered from a 8 mg crude starting material) while immunoblot analysis confirmed immunological reactivity of the purified proteins. Such a purification method is rapid, inexpensive, and efficient in generating proteins of sufficient purity for use in monoclonal antibody (mAb) production, protein sequencing and general molecular, immunological, and cellular studies.

Development of a capillary electrophoresis method for the simultaneous analysis of artificial sweeteners, preservatives and colours in soft drinks

A rapid capillary electrophoresis method was developed simultaneously to determine artificial sweeteners, preservatives and colours used as additives in carbonated soft drinks. Resolution between all additives occurring together in soft drinks was successfully achieved within a 15-min run-time by employing the micellar electrokinetic chromatography mode with a 20 mM carbonate buffer at pH 9.5 as the aqueous phase and 62 mM sodium dodecyl sulfate as the micellar phase. By using a diode-array detector to monitor the UV-visible range (190-600 nm), the identity of sample components, suggested by migration time, could be confirmed by spectral matching relative to standards.

Development of a fast capillary electrophoresis method to determine inorganic cations in biodiesel samples

The aim of this study was to develop a fast capillary electrophoresis method for the determination of inorganic cations (Na(+), K(+), Ca(2+), Mg(2+)) in biodiesel samples, using barium (Ba(2+)) as the internal standard. The running electrolyte was optimized through effective mobility curves in order to select the co-ion and Peakmaster software was used to determine electromigration dispersion and buffer capacity. The optimum background electrolyte was composed of 10 mmol L(-1) imidazole and 40 mmol L(-1) of acetic acid. Separation was conducted in a fused-silica capillary (32 cm total length and 23.5 cm effective length, 50 mu m I.D.), with indirect UV detection at 214 nm. The migration time was only 36 s. In order to obtain the optimized conditions for extraction, a fractional factorial experimental design was used. The variables investigated were biodiesel mass, pH, extractant volume, agitation and sonication time. The optimum conditions were: biodiesel mass of 200 mg, extractant volume of 200 mu L. and agitation of 20 min. The method is characterized by good linearity in the concentration range of 0.5-20 mg kg(-1) (r > 0.999), limit of detection was equal to 0.3 mg kg(-1), inter-day precision was equal to 1.88% and recovery in the range of 88.0-120%. The developed method was successfully applied to the determination of cations in biodiesel samples. (c) 2010 Elsevier B.V. All rights reserved.

Development of a fast capillary electrophoresis method for the determination of propranolol-Total analysis time reduction strategies

The aim of this study was to develop a fast capillary electrophoresis method for the determination of propranolol in pharmaceutical preparations. In the method development the pH and constituents of the background electrolyte were selected using the effective mobility versus pH curves. Benzylamine was used as the internal standard. The background electrolyte was composed of 60 mmol L(-1) tris(hydroxymethyl)aminomethane and 30 mmol L(-1) 2-hydroxyisobutyric acid,at pH 8.1. Separation was conducted in a fused-silica capillary (32 cm total length and 8.5 cm effective length, 50 mu m I.D.) with a short-end injection configuration and direct UV detection at 214 nm. The run time was only 14 s. Three different strategies were studied in order to develop a fast CE method with low total analysis time for propranolol analysis: low flush time (Lflush) 35 runs/h, without flush (Wflush) 52 runs/h, and Invert (switched polarity) 45 runs/h. Since the three strategies developed are statistically equivalent, Mush was selected due to the higher analytical frequency in comparison with the other methods. A few figures of merit of the proposed method include: good linearity (R(2) > 0.9999); limit of detection of 0.5 mg L(-1): inter-day precision better than 1.03% (n = 9) and recovery in the range of 95.1-104.5%. (C) 2009 Elsevier B.V. All rights reserved.

Validation of a stability indicating capillary electrophoresis method for the determination of darunavir in tablets and comparison with the of infrared absorption spectroscopic method

Darunavir (DRV) is a protease inhibitor used in the treatment of HIV infection, which constitutes a keystone in the therapy of patients infected with this virus. There is no monograph described in official compendia. The literature provides few methods of analysis for the determination of DRV in pharmaceuticals which include TLC, IR, UPLC, HPLC, HPLC-MS, HPLC-MS/MS, but there are no reports of the use of capillary electrophoresis (CE) for the determination of this drug. Thus, this research proposed the development and validation of a CE method for the determination of DRV in tablets. The method was completely validated according to the International Conference on Harmonization guidelines, showing linearity, selectivity, precision, accuracy and robustness. The migration was achieved in less than 1 minute using fused-silica uncoated capillary with an id of 50 μm and total length of 21 cm and voltage of +20 kV. The sample injection was performed in the hydrodynamic mode. The method was linear over the concentration range of 50-200 μg mL-1 with correlation coefficient 0.9998 and limits of detection and quantification of 7.29 and 22.09 μg mL-1, respectively. The drug was subjected to acid, base, oxidation and photolysis degradation. Degradation products were found interfering with the assay of DRV, therefore the method can be regarded as stability indicating. The validated method is useful and appropriate for the routine quality control of DRV in tablets.

DNA-protein binding assays from a single sea urchin egg: a high-sensitivity capillary electrophoresis method.

A capillary electrophoresis method has been developed to study DNA-protein complexes by mobility-shift assay. This method is at least 100 times more sensitive than conventional gel mobility-shift procedures. Key features of the technique include the use of a neutral coated capillary, a small amount of linear polymer in the separation medium, and use of covalently dye-labeled DNA probes that can be detected with a commercially available laser-induced fluorescence monitor. The capillary method provides quantitative data in runs requiring < 20 min, from which dissociation constants are readily determined. As a test case we studied interactions of a developmentally important sea urchin embryo transcription factor, SpP3A2. As little as 2-10 x 10(6) molecules of specific SpP3A2-oligonucleotide complex were reproducibly detected, using recombinant SpP3A2, crude nuclear extract, egg lysates, and even a single sea urchin egg lysed within the capillary column.

Determination of sorbate and benzoate in beverage samples by capillary electrophoresis - Optimization of the method with inspection of ionic mobilities

The aim of this study was to develop a fast capillary electrophoresis method for the determination of benzoate and sorbate ions in commercial beverages. In the method development the pH and constituents of the background electrolyte were selected using the effective mobility versus pH curves. As the high resolution obtained experimentally for sorbate and benzoate in the studies presented in the literature is not in agreement with that expected from the ionic mobility values published, a procedure to determine these values was carried out. The salicylate ion was used as the internal standard. The background electrolyte was composed of 25 mmol L(-1) tris(hydroxymethyl)aminomethane and 12.5 mmol L(-1) 2-hydroxyisobutyric acid, atpH 8.1.Separation was conducted in a fused-silica capillary(32 cm total length and 8.5 cm effective length, 50 mu m I.D.), with short-end injection configuration and direct UV detection at 200 nm for benzoate and salicylate and 254 nm for sorbate ions. The run time was only 28 s. A few figures of merit of the proposed method include: good linearity (R(2) > 0.999), limit of detection of 0.9 and 0.3 mg L(-1) for benzoate and sorbate, respectively, inter-day precision better than 2.7% (n =9) and recovery in the range 97.9-105%. Beverage samples were prepared by simple dilution with deionized water (1:11, v/v). Concentrations in the range of 197-401 mg L(-1) for benzoate and 28-144 mg L(-1) for sorbate were found in soft drinks and tea. (c) 2008 Elsevier B.V. All rights reserved.

Rapid method for the determination. of organic acids in wine by capillary electrophoresis with indirect UV detection

A capillary electrophoresis method for organic acids in wine was developed and validated. The optimal electrolyte consisted of 10 mmol/L 3,5-dinitrobenzoic acid (DNB) at pH 3.6 containing 0.2 mmol/L cetyltrimethylammonium bromide as flow reverser. DNB was chosen because it has an effective mobility similar to the organic acids under investigation, good buffering capacity at pH 3.6, and good chromophoric characteristics for indirect UV-absorbance detection at 254 nm. Sample preparation involved dilution and filtration. The method showed good performance characteristics: Linearity at 6 to 285 mg/L (r > 0.99); detection and quantification limits of 0.64 to 1.55 and 2.12 to 5.15 mg/L, respectively; separation time of less than 5.5 min. Coefficients of variation for ten injections were less than 5% and recoveries varied from 95% to 102%. Application to 23 samples of Brazilian wine confirmed good repeatability and demonstrated wide variation in the organic acid concentrations. (C) 2008 Elsevier Ltd. All rights reserved.

Analysis of quinolizidine alkaloids in Sophora flavescens Ait. by capillary electrophoresis with tris(2,2 '-bipyridyl) ruthenium (II)-based electrochemiluminescence detection

A capillary electrophoresis method coupled with electrochemiluminescence detection for the analysis of quinolizidine alkaloids was established, especially, oxymatrine (OMT) which could not be measured by previous electrochemiluminescence methods was detected sensitively herein. Complete separation of sophoridine (SR), matrine (MT) and OMT was achieved within 13 min using a background electrolyte of 50mM phosphate buffer at pH 8.4 and a separation voltage of 15 kV. The calibration curves showed a linear range from 2.8 x 10(-8) to 4.4 x 10(-7) M for SR, 2.7 x 10(-8) to 4.4 x 10(-7) M for MT.

Analysis of five pharmacologically active compounds from Rhodiola for natural product drug discovery with capillary electrophoresis

A rapid capillary electrophoresis method for the separation of five natural pharmacologically active compounds from extracted Rhodiola, namely salidroside, tyrosol, rhodionin, gallic acid and ethyl gallate has been developed. The separation of five natural pharmacologically active compounds was carried out in a fused-silica capillary with 14 mM boric acid, 30 mM SDS and 2.5% acetonitrile, adjusted to pH 10.7 with NaOH. Applied potential was 21 kV. The temperature of the capillary was maintained at 25 degreesC by the instrument thermostating system, with the correlation coefficients of 0.9805-0.9989 for migration time, and relative standards of < 3.52% for peak areas. The established method is rapid and reproducible for the separation of five natural pharmacologically compounds from extracts of Rhodiola with satisfactory results.

Rapid authentication of ginseng species using microchip electrophoresis with laser-induced fluorescence detection

Ginseng is one of the most expensive Chinese herbal medicines and the effectiveness of ginseng depends strongly on its botanical sources and the use of different parts of the plants. In this study, a microchip electrophoresis method coupled with the polymerase chain reaction (PCR)-short tandem repeats (STR) technique was developed for rapid authentication of ginseng species. A low viscosity hydroxypropyl methylcellulose (HPMC) solution was used as the sieving matrix for separation of the amplified STR fragments. The allele sizing of the amplified PCR products could be detected within 240 s or less. Good reproducibility and accuracy of the fragment size were obtained with the relative standard deviation for the allele sizes less than 1.0% (n = 11). At two microsatellite loci (CT 12, CA 33), American ginseng had a different allele pattern on the electropherograms compared with that of the Oriental ginseng. Moreover, cultivated and wild American ginseng can be distinguished on the basis of allele sizing. This work establishes the feasibility of fast genetic authentication of ginseng species by use of microchip electrophoresis.

Simultaneous and ultrarapid determination of reactive oxygen species and reduced glutathione in apoptotic leukemia cells by microchip electrophoresis

A microchip electrophoresis method coupled with laser-induced fluorescence (LIF) detection was established for simultaneous determination of two kinds of intracellular signaling molecules (reactive oxygen species, ROS, and reduced glutathione, GSH) related to apoptosis and oxidative stress. As the probe dihydrorhodamine-123 (DHR123) can be converted intracellularly by ROS to the fluorescent rhodamine-123 (Rh123), and the probe naphthalene-2,3-dicarboxaldehyde (NDA) can react quickly with GSH to produce a fluorescent adduct, rapid determination of Rh-123 and GSH was achieved on a glass microchip within 27 s using a 20 mm borate buffer (pH 9.2). The established method was tested to measure the intracellular ROS and GSH levels in acute promyelocytic leukemia (APL)-derived NB4 cells. An elevation of intracellular ROS and depletion of GSH were observed in apoptotic N134 cells induced by arsenic trioxide (AS(2)O(3)) at low concentration (1-2 mu m). Buthionine sulfoximine (BSO), in combination with AS(2)O(3) enhanced the decrease of reduced GSH to a great extent. The combined treatment of AS(2)O(3) and hydrogen peroxide (H2O2) led to an inverse relationship between the concentrations of ROS and GSH obtained, showing the proposed method can readily evaluate the generation of ROS, which occurs simultaneously with the consumption of the inherent antioxidant.

Determination of dissociation constants of Strychnos alkaloids from Strychnos nux-vomica L. by capillary electrophoresis

An accurate capillary electrophoresis method was developed for the determination of dissociation constants of five Strychnos alkaloids from Strychnos nux-vomica L. The method relies on measuring the effective mobility of the solute as a function of the buffer pH. The mathematical relationship was strictly derived from the fundamental electrophoresis theory and the dissociation equilibrium of a weak base without any simplifications. Careful optimization of the running buffer permitted base-line resolution of the five structurally similar alkaloids.

Influence of the buffer environment on the Relative Biological Effectiveness of carbon ion radiation, investigated by Scanning Force Microscopy and gel electrophoresis

This work focuses on the analysis of the influence of environment on the relative biological effectiveness (RBE) of carbon ions on molecular level. Due to the high relevance of RBE for medical applications, such as tumor therapy, and radiation protection in space, DNA damages have been investigated in order to understand the biological efficiency of heavy ion radiation. The contribution of this study to the radiobiology research consists in the analysis of plasmid DNA damages induced by carbon ion radiation in biochemical buffer environments, as well as in the calculation of the RBE of carbon ions on DNA level by mean of scanning force microscopy (SFM). In order to study the DNA damages, besides the common electrophoresis method, a new approach has been developed by using SFM. The latter method allows direct visualisation and measurement of individual DNA fragments with an accuracy of several nanometres. In addition, comparison of the results obtained by SFM and agarose gel electrophoresis methods has been performed in the present study. Sparsely ionising radiation, such as X-rays, and densely ionising radiation, such as carbon ions, have been used to irradiate plasmid DNA in trishydroxymethylaminomethane (Tris buffer) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES buffer) environments. These buffer environments exhibit different scavenging capacities for hydroxyl radical (HO0), which is produced by ionisation of water and plays the major role in the indirect DNA damage processes. Fragment distributions have been measured by SFM over a large length range, and as expected, a significantly higher degree of DNA damages was observed for increasing dose. Also a higher amount of double-strand breaks (DSBs) was observed after irradiation with carbon ions compared to X-ray irradiation. The results obtained from SFM measurements show that both types of radiation induce multiple fragmentation of the plasmid DNA in the dose range from D = 250 Gy to D = 1500 Gy. Using Tris environments at two different concentrations, a decrease of the relative biological effectiveness with the rise of Tris concentration was observed. This demonstrates the radioprotective behavior of the Tris buffer solution. In contrast, a lower scavenging capacity for all other free radicals and ions, produced by the ionisation of water, was registered in the case of HEPES buffer compared to Tris solution. This is reflected in the higher RBE values deduced from SFM and gel electrophoresis measurements after irradiation of the plasmid DNA in 20 mM HEPES environment compared to 92 mM Tris solution. These results show that HEPES and Tris environments play a major role on preventing the indirect DNA damages induced by ionising radiation and on the relative biological effectiveness of heavy ion radiation. In general, the RBE calculated from the SFM measurements presents higher values compared to gel electrophoresis data, for plasmids irradiated in all environments. Using a large set of data, obtained from the SFM measurements, it was possible to calculate the survive rate over a larger range, from 88% to 98%, while for gel electrophoresis measurements the survive rates have been calculated only for values between 96% and 99%. While the gel electrophoresis measurements provide information only about the percentage of plasmids DNA that suffered a single DSB, SFM can count the small plasmid fragments produced by multiple DSBs induced in a single plasmid. Consequently, SFM generates more detailed information regarding the amount of the induced DSBs compared to gel electrophoresis, and therefore, RBE can be calculated with more accuracy. Thus, SFM has been proven to be a more precise method to characterize on molecular level the DNA damage induced by ionizing radiations.

Determination of polyamines in organic extracts from roots of Canavalia ensiformis by capillary electrophoresis

A rapid, selective and specific capillary zone electrophoresis method to determine polyamines in organic extracts from roots of Canavalia ensiformis (Jack Beans) was developed using ultra violet (UV) detection. Canavalia ensiformis is relatively free from diseases and it is used as reference in allelopathy studies. Polyamines are widely distributed in plant and it could be involved in plant pathogen interactions. Optimal separation was achieved using 15 mmol.L-1formic acid (pH 3.0) + 4 mmol.L-1 imidazole as a background electrolyte. It was possible to identify and quantify the polyamines on herbal samples in the presence of other phytochemical substances and analyze them quickly (up to 6 min). The applicability of this method was evaluated in crude organic extracts from roots of Canavalia ensiformis.