The Experts below are selected from a list of 18339 Experts worldwide ranked by ideXlab platform
Janusz Pawliszyn - One of the best experts on this subject based on the ideXlab platform.
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development of a simple ampholyte free Isoelectric Focusing slab electrophoresis for protein fractionation
Journal of Chromatography A, 2009Co-Authors: Yanwei Zhan, Tibebe Lemma, Marcel Musteata, Janusz PawliszynAbstract:Abstract Sample preparation is often necessary to separate and concentrate various compounds prior to analysis of complex samples. In this regard, Isoelectric Focusing (IEF) is one of the best sample preparation methods. With this approach, however, carrier ampholytes have to be introduced into the samples, which may result in matrix interferences. In this paper, a simple ampholyte-free IEF free-flow electrophoresis design was developed for the separation of proteins. β-Lactoglobulin, hemoglobin, myoglobin and cytochrome c were selected as model analytes. The experimental design took advantage of the electrolysis-driven production of H+ and OH− ions that migrated from the anode and cathode, respectively, establishing a pH gradient spanning from 2.3 to 8.9. The separation chamber was filled with silanized glass beads as a support medium. Dialysis membranes were mounted at the two sides of the separation chamber (made of glass slides) and sealed with 2% agarose gel. The separated proteins drained from the outlets of the separation chamber and could be successfully collected into small glass tubes. The Focusing process was visually observed and the separation was confirmed by capillary Isoelectric Focusing (cIEF) with pI markers.
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Dynamic kinetic capillary Isoelectric Focusing : A powerful tool for studying protein-DNA interactions
Analytical Chemistry, 2007Co-Authors: Zhen Liu, Andrei P. Drabovich, Sergey N. Krylov, Janusz PawliszynAbstract:A new method called dynamic kinetic capillary Isoelectric Focusing (DK-CIEF) is presented for the study of protein−DNA interactions. The method is based on CIEF with laser-induced fluorescence-whole column imaging detection in which protein−DNA complexes are separated with spatial resolution while dissociations of the complexes are dynamically monitored using a CCD camera with temporal resolution. This method allows for the discrimination of different complexes and the measurement of the individual dissociation rate constants.
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capillary Isoelectric Focusing with whole column imaging detection for analysis of proteins and peptides
Journal of Biochemical and Biophysical Methods, 1999Co-Authors: Qinglu Mao, Janusz PawliszynAbstract:Abstract Whole column imaging detection has been developed for capillary Isoelectric Focusing (CIEF) of proteins and peptides. In this imaged CIEF technique, a solution of sample and ampholytes was introduced into a short (4–5 cm), internally coated capillary stabilized by a cartridge. After applying high DC voltage, the Isoelectric Focusing process takes place and the focused zones are monitored in a real-time mode using the imaging detectors developed. Three types of imaging detectors have been developed including refractive index gradient, laser-induced fluorescence (LIF), and absorption. Of these, absorption imaging detection is the most practical at the present time due to its quantitative ability and universal characteristics. Whole column imaging detection eliminates the mobilization step required for single point detection after the Focusing process. Therefore, it provides a fast analysis speed (3–5 min for each sample), and avoids the disadvantages associated with the mobilization process, such as distortion of pH gradient and loss in resolution. In this paper, we review the methodology of imaged CIEF as well as progress in instrumental development, IEF performed on a microchip, and the application to protein and peptide analysis.
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Dual Detection for Capillary Isoelectric Focusing with Refractive Index Gradient and Absorption Imaging Detectors
Analytical Chemistry, 1994Co-Authors: Janusz PawliszynAbstract:A unique imaging detector was constructed for capillary Isoelectric Focusing (CIEF) by combining a selective optical absorption imaging detector with a universal refractive index gradient imaging detector. All sample zones focused inside a 100-μm-i.d., 4-cm-long capillary by Isoelectric Focusing were detected in a real-time fashion by the universal imaging detector, and their spectroscopic properties were measured by the absorption imaging detector almost simultaneously. The refractive index gradient imaging is based on a dark-field Toepler-Schlieren system
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ampholyte free Isoelectric Focusing of proteins in cone shaped capillaries
Journal of Microcolumn Separations, 1993Co-Authors: Janusz PawliszynAbstract:A simple method of generating pH gradients for use in capillary Isoelectric Focusing was described and evaluated experimentally. In the proposed scheme, the cone shaped capillary filled with a conventional buffer is used to produce an effect of nonuniform heating which then generate temperature and corresponding pH gradients along the capillary axis. Concentration and separation of proteins by applying a simple Isoelectric Focusing system based on this principle is demonstrated. An inexpensive absorption imaging detection system is used to continuously monitor progress of the separation and concentration processes.
Richard B M Schasfoort - One of the best experts on this subject based on the ideXlab platform.
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microfluidic high resolution free flow Isoelectric Focusing
Analytical Chemistry, 2007Co-Authors: Dietrich Kohlheyer, Jan C T Eijkel, Stefan Schlautmann, And Albert Van Den Berg, Richard B M SchasfoortAbstract:A microfluidic free-flow Isoelectric Focusing glass chip for separation of proteins is described. Free-flow Isoelectric Focusing is demonstrated with a set of fluorescent standards covering a wide range of Isoelectric points from pH 3 to 10 as well as the protein HSA. With respect to an earlier developed device, an improved microfluidic FFE chip was developed. The improvements included the usage of multiple sheath flows and the introduction of preseparated ampholytes. Preseparated ampholytes are commonly used in large-scale conventional free-flow Isoelectric Focusing instruments but have not been used in micromachined devices yet. Furthermore, the channel depth was further decreased. These adaptations led to a higher separation resolution and peak capacity, which were not achieved with previously published free-flow Isoelectric Focusing chips. An almost linear pH gradient ranging from pH 2.5 to 11.5 between 1.2 and 2 mm wide was generated. Seven Isoelectric Focusing markers were successfully and clearly separated within a residence time of 2.5 s and an electrical field of 20 V mm-1. Experiments with pI markers proved that the device is fully capable of separating analytes with a minimum difference in Isoelectric point of ∆(pI) = 0.4. Furthermore, the results indicate that even a better resolution can be achieved. The theoretical minimum difference in Isoelectric point is ∆(pI) = 0.23 resulting in a peak capacity of 29 peaks within 1.8 mm. This is an 8-fold increase in peak capacity to previously published results. The Focusing of pI markers led to an increase in concentration by factor 20 and higher. Further improvement in terms of resolution seems possible, for which we envisage that the influence of electroosmotic flow has to be further reduced. The performance of the microfluidic free-flow Isoelectric Focusing device will enable new applications, as this device might be used in clinical analysis where often low sample volumes are available and fast separation times are essential.
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free flow zone electrophoresis and Isoelectric Focusing using a microfabricated glass device with ion permeable membranes
Lab on a Chip, 2006Co-Authors: Dietrich Kohlheyer, Stefan Schlautmann, G A J Besselink, Richard B M SchasfoortAbstract:This paper describes a microfabricated free-flow electrophoresis device with integrated ion permeable membranes. In order to obtain continuous lanes of separated components an electrical field is applied perpendicular to the sample flow direction. This sample stream is sandwiched between two sheath flow streams, by hydrodynamic Focusing. The separation chamber has two open side beds with inserted electrodes to allow ventilation of gas generated during electrolysis. To hydrodynamically isolate the separation compartment from the side electrodes, a photo-polymerizable monomer solution is exposed to UV light through a slit mask for in situ membrane formation. These so-called salt-bridges resist the pressure driven fluid, but allow ion transport to enable electrical connection. In earlier devices the same was achieved by using open side channel arrays. However, only a small fraction of the applied voltage was effectively utilized across the separation chamber during free-flow electrophoresis and free-flow Isoelectric Focusing. Furthermore, the spreading of the carrier ampholytes into the side channels resulted in a very restricted pH gradient inside the separation chamber. The chip presented here allows at least 10 times more efficient use of the applied potential and a nearly linear pH gradient from pH 3 to 10 during free-flow Isoelectric Focusing could be established. Furthermore, the application of hydrodynamic Focusing in combination with free-flow electrophoresis can be used for guiding the separated components to specific chip outlets. As a demonstration, several standard fluorescent markers were separated and focused by free-flow zone electrophoresis and by free-flow Isoelectric Focusing employing a transversal voltage of up to 150 V across the separation chamber.
Dietrich Kohlheyer - One of the best experts on this subject based on the ideXlab platform.
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microfluidic high resolution free flow Isoelectric Focusing
Analytical Chemistry, 2007Co-Authors: Dietrich Kohlheyer, Jan C T Eijkel, Stefan Schlautmann, And Albert Van Den Berg, Richard B M SchasfoortAbstract:A microfluidic free-flow Isoelectric Focusing glass chip for separation of proteins is described. Free-flow Isoelectric Focusing is demonstrated with a set of fluorescent standards covering a wide range of Isoelectric points from pH 3 to 10 as well as the protein HSA. With respect to an earlier developed device, an improved microfluidic FFE chip was developed. The improvements included the usage of multiple sheath flows and the introduction of preseparated ampholytes. Preseparated ampholytes are commonly used in large-scale conventional free-flow Isoelectric Focusing instruments but have not been used in micromachined devices yet. Furthermore, the channel depth was further decreased. These adaptations led to a higher separation resolution and peak capacity, which were not achieved with previously published free-flow Isoelectric Focusing chips. An almost linear pH gradient ranging from pH 2.5 to 11.5 between 1.2 and 2 mm wide was generated. Seven Isoelectric Focusing markers were successfully and clearly separated within a residence time of 2.5 s and an electrical field of 20 V mm-1. Experiments with pI markers proved that the device is fully capable of separating analytes with a minimum difference in Isoelectric point of ∆(pI) = 0.4. Furthermore, the results indicate that even a better resolution can be achieved. The theoretical minimum difference in Isoelectric point is ∆(pI) = 0.23 resulting in a peak capacity of 29 peaks within 1.8 mm. This is an 8-fold increase in peak capacity to previously published results. The Focusing of pI markers led to an increase in concentration by factor 20 and higher. Further improvement in terms of resolution seems possible, for which we envisage that the influence of electroosmotic flow has to be further reduced. The performance of the microfluidic free-flow Isoelectric Focusing device will enable new applications, as this device might be used in clinical analysis where often low sample volumes are available and fast separation times are essential.
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free flow zone electrophoresis and Isoelectric Focusing using a microfabricated glass device with ion permeable membranes
Lab on a Chip, 2006Co-Authors: Dietrich Kohlheyer, Stefan Schlautmann, G A J Besselink, Richard B M SchasfoortAbstract:This paper describes a microfabricated free-flow electrophoresis device with integrated ion permeable membranes. In order to obtain continuous lanes of separated components an electrical field is applied perpendicular to the sample flow direction. This sample stream is sandwiched between two sheath flow streams, by hydrodynamic Focusing. The separation chamber has two open side beds with inserted electrodes to allow ventilation of gas generated during electrolysis. To hydrodynamically isolate the separation compartment from the side electrodes, a photo-polymerizable monomer solution is exposed to UV light through a slit mask for in situ membrane formation. These so-called salt-bridges resist the pressure driven fluid, but allow ion transport to enable electrical connection. In earlier devices the same was achieved by using open side channel arrays. However, only a small fraction of the applied voltage was effectively utilized across the separation chamber during free-flow electrophoresis and free-flow Isoelectric Focusing. Furthermore, the spreading of the carrier ampholytes into the side channels resulted in a very restricted pH gradient inside the separation chamber. The chip presented here allows at least 10 times more efficient use of the applied potential and a nearly linear pH gradient from pH 3 to 10 during free-flow Isoelectric Focusing could be established. Furthermore, the application of hydrodynamic Focusing in combination with free-flow electrophoresis can be used for guiding the separated components to specific chip outlets. As a demonstration, several standard fluorescent markers were separated and focused by free-flow zone electrophoresis and by free-flow Isoelectric Focusing employing a transversal voltage of up to 150 V across the separation chamber.
Eliska Glovinova - One of the best experts on this subject based on the ideXlab platform.
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analytical aspects of carrier ampholyte free Isoelectric Focusing
Journal of Chromatography A, 2001Co-Authors: Jan Pospichal, Eliska GlovinovaAbstract:The applicability of carrier ampholyte-free Isoelectric Focusing (CAF-IEF) for analyses of ampholytes is demonstrated. The suggested method is based on the principle of both side regulated ionic matrix in CAF-IEF. A sharp step of pH is created in the column filled with a sample dissolved in a background electrolyte by influence of current and solvolytic fluxes. Here, ampholytes are focused upon. The magnitude of the step, its velocity and direction of its movement can be regulated electrically. In this manner, favorable separation properties of the system can be set up, even during the run. This brings several advantages over conventional methods. The principles of the separation can be easily changed, permitting selective pre-concentration (trapping) of minor components by processing large amounts of a sample to be preformed, effective isotachophoresis or IEF pre-separation and final electrophoretic analysis in one run. Advantages of these combinations are discussed together with the right choice of the working electrolyte. A 1000-fold increase in amount of substance in a column can be achieved for both isotachophoresis and capillary zone electrophoresis combined with CAF-IEF pre-concentration at reasonable working conditions. It enables a limit of detection at the nmol/l level with a concentration factor of about 10(7) to be reached.
Fuquan Yang - One of the best experts on this subject based on the ideXlab platform.
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carrier ampholyte free Isoelectric Focusing on a paper based analytical device for the fractionation of proteins
Journal of Separation Science, 2018Co-Authors: Songfang Xie, Han Gao, Lili Niu, Zhensheng Xie, Fang Fang, Fuquan YangAbstract:Isoelectric Focusing plays a critical role in the analysis of complex protein samples. Conventionally, Isoelectric Focusing is implemented with carrier ampholytes in capillary or immobilized pH gradient gel. In this study, we successfully exhibited a carrier ampholyte-free Isoelectric Focusing on paper-based analytical device. Proof of the concept was visually demonstrated with color model proteins. Experimental results showed that not only a pH gradient was well established along the open paper fluidic channel as confirmed by pH indicator strip, the pH gradient range could also be tuned by the catholyte or anolyte. Furthermore, the Isoelectric Focusing fractions from the paper channel can be directly cut and recovered into solutions for post analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. This paper-based Isoelectric Focusing method is fast, cheap, simple and easy to operate, and could potentially be used as a cost-effective protein sample clean-up method for target protein analysis with mass spectrometry.
