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Sergey N Krylov - One of the best experts on this subject based on the ideXlab platform.
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Empirical predictor of conditions that support ideal‐filter capillary electrophoresis
Electrophoresis, 2020Co-Authors: Tong Ye Wang, Sergey N KrylovAbstract:Ideal-filter CE (IFCE) is a method for the selection of affinity binders for protein targets from oligonucleotide libraries, for example, random-sequence oligonucleotide libraries and DNA-encoded libraries, in a single step of partitioning. In IFCE, protein-oligonucleotide complexes and unbound oligonucleotides move in the opposite directions, facilitating very high efficiency of their partitioning. For any given protein target and oligonucleotide library, protein-oligonucleotide complexes and unbound oligonucleotides move in the opposite directions only for a limited range of EOF mobilities, which, in turn, corresponds to a limited range of pH and ionic strength values of the Running Buffer. Rational design of IFCE-based partitioning requires a priori knowledge of this range of pH and ionic strength values, and here we introduce an approach to predict this range for a given type of the Running Buffer. The approach involves measuring EOF mobilities for a relatively wide range of pH and ionic strength (I) values and finding an empirical predictor function that related the EOF mobility with pH and ionic strength. In this work, we developed a predictor function for a Running Buffer (Tris-HCl) that is commonly used in CE-based partitioning of affinity binders for protein targets. This predictor function can be immediately used for the rational design of IFCE-based partitioning in this Running Buffer, while the described approach will be used to develop predictor functions for other types of Running Buffer if needed.
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empirical predictor of conditions that support ideal filter capillary electrophoresis
Electrophoresis, 2020Co-Authors: Tong Ye Wang, Liang Hu, Sergey N KrylovAbstract:: Ideal-filter CE (IFCE) is a method for the selection of affinity binders for protein targets from oligonucleotide libraries, for example, random-sequence oligonucleotide libraries and DNA-encoded libraries, in a single step of partitioning. In IFCE, protein-oligonucleotide complexes and unbound oligonucleotides move in the opposite directions, facilitating very high efficiency of their partitioning. For any given protein target and oligonucleotide library, protein-oligonucleotide complexes and unbound oligonucleotides move in the opposite directions only for a limited range of EOF mobilities, which, in turn, corresponds to a limited range of pH and ionic strength values of the Running Buffer. Rational design of IFCE-based partitioning requires a priori knowledge of this range of pH and ionic strength values, and here we introduce an approach to predict this range for a given type of the Running Buffer. The approach involves measuring EOF mobilities for a relatively wide range of pH and ionic strength (I) values and finding an empirical predictor function that related the EOF mobility with pH and ionic strength. In this work, we developed a predictor function for a Running Buffer (Tris-HCl) that is commonly used in CE-based partitioning of affinity binders for protein targets. This predictor function can be immediately used for the rational design of IFCE-based partitioning in this Running Buffer, while the described approach will be used to develop predictor functions for other types of Running Buffer if needed.
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Direct Quantitative Analysis of Multiple microRNAs (DQAMmiR) with Peptide Nucleic Acid Hybridization Probes
Analytical chemistry, 2018Co-Authors: Mansi Anand, Svetlana M. Krylova, Burton B. Yang, Stanley K. Liu, George M. Yousef, Sergey N KrylovAbstract:Direct quantitative analysis of multiple miRNAs (DQAMmiR) is a hybridization-based assay, in which the excess of the DNA hybridization probes is separated from the miRNA-probe hybrids, and the hybrids are separated from each other in gel-free capillary electrophoresis (CE) using two types of mobility shifters: single-strand DNA binding protein (SSB) added to the CE Running Buffer and peptide drag tags conjugated with the probes. Here we introduce the second-generation DQAMmiR, which utilizes peptide nucleic acid (PNA) rather than DNA hybridization probes and requires no SSB in the CE Running Buffer. PNA probes are electrically neutral, while PNA–miRNA hybrids are negatively charged, and this difference in charge can be a basis for separation of the hybrids from the probes. In this proof-of-principle work, we first experimentally confirmed that the PNA–RNA hybrid was separable from the excess of the PNA probe without SSB in the Running Buffer, resulting in a near 10 min time window, which would allow, theo...
Bert C. Lynn - One of the best experts on this subject based on the ideXlab platform.
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A novel Bicine Running Buffer system for doubled sodium dodecyl sulfate – polyacrylamide gel electrophoresis of membrane proteins
Electrophoresis, 2006Co-Authors: Taufika Islam Williams, Jennifer C. Combs, Anup P. Thakur, Herbert J. Strobel, Bert C. LynnAbstract:A novel, Bicine-based SDS-PAGE Buffer system was developed for the analysis of membrane proteins. The method involves molecular weight-based separations of fully denatured and solubilized proteins in two dimensions. This doubled SDS-PAGE (dSDS-PAGE) approach produced a diagonal arrangement of protein spots and successfully circumvented problems associated with membrane proteome analysis involving traditional gel-based methods. Membrane proteins from the anaerobic bacterium Clostridium thermocellum were used for these investigations. Tricine-dSDS-PAGE and the newly developed Bicine-dSDS-PAGE were compared with the standard glycine-dSDS-PAGE (Laemmli protocol) in their suitability to separate C. thermocellum membrane proteins. Large-format gel experiments using optimized gel preparation and Running Buffer conditions revealed a 112% increase in protein spot count for Tricine-dSDS-PAGE and a 151% increase for Bicine-dSDS-PAGE, compared to glycine-dSDS-PAGE. The data clearly indicated that Bicine-dSDS-PAGE is a superior method for the analysis of membrane proteins, providing enhanced resolution and protein representation.
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a novel bicine Running Buffer system for doubled sodium dodecyl sulfate polyacrylamide gel electrophoresis of membrane proteins
Electrophoresis, 2006Co-Authors: Taufika Islam Williams, Jennifer C. Combs, Anup P. Thakur, Herbert J. Strobel, Bert C. LynnAbstract:A novel, Bicine-based SDS-PAGE Buffer system was developed for the analysis of membrane proteins. The method involves molecular weight-based separations of fully denatured and solubilized proteins in two dimensions. This doubled SDS-PAGE (dSDS-PAGE) approach produced a diagonal arrangement of protein spots and successfully circumvented problems associated with membrane proteome analysis involving traditional gel-based methods. Membrane proteins from the anaerobic bacterium Clostridium thermocellum were used for these investigations. Tricine-dSDS-PAGE and the newly developed Bicine-dSDS-PAGE were compared with the standard glycine-dSDS-PAGE (Laemmli protocol) in their suitability to separate C. thermocellum membrane proteins. Large-format gel experiments using optimized gel preparation and Running Buffer conditions revealed a 112% increase in protein spot count for Tricine-dSDS-PAGE and a 151% increase for Bicine-dSDS-PAGE, compared to glycine-dSDS-PAGE. The data clearly indicated that Bicine-dSDS-PAGE is a superior method for the analysis of membrane proteins, providing enhanced resolution and protein representation.
Yuzhi Fang - One of the best experts on this subject based on the ideXlab platform.
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Simultaneous determination of flavonoids and anthraquinones in chrysanthemum by capillary electrophoresis with amperometry detection
Chinese Chemical Letters, 2010Co-Authors: Yin Yan Zhang, Zhiyong Yang, Qingjiang Wang, Jin Kun Zhu, Yuzhi FangAbstract:Abstract A high-performance capillary electrophoresis with amperometry detection method (CE-AD) has been developed for the analysis of flavonoids and anthraquinones (emodin, kaempferol, apigenin, luteolin and rhein) in chrysanthemum. Under optimum conditions, these five analytes were base-line separated within 17 min using a borate–phosphate Running Buffer (1.5 × 10−2 mol/L borate–3 × 10−2 mol/L phosphate Running Buffer, pH 9.0) at a working potential of +0.90 V (vs. SCE) and a separation voltage of 19 kV. The linear relationship between concentration and current response was obtained with detection limits (S/N = 3) ranging from 1.0 × 10−7 to 2.1 × 10−7 g/mL for all analytes. This proposed method was successfully used in the analysis of four kinds of chrysanthemum with relatively simple extraction procedures, the assay results were satisfactory.
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Simultaneous determination of flavonoids and phenolic acids in Chinese herbal tea by beta-cyclodextrin based capillary zone electrophoresis
Mikrochimica Acta, 2009Co-Authors: Langzhu Chi, Shuqing Dong, Qingjiang Wang, Yuzhi FangAbstract:Kaempferol, apigenin, rutin, quercetin, luteolin and ferulic acid are separated and detected in Chinese herbal tea using capillary zone electrophoresis coupled to amperometric detection. The phosphate Running Buffer also contains s-cyclodextrin (s-CD), which assists in separation and gives excellent separations within 20 min and detection limits as low as 10 ng mL−1 (S/N = 3). The effects of working electrode potential, pH and concentration of Running Buffer, concentration of s-CD, separation voltage and injection time were investigated. The method was applied to analyze tea samples with recoveries in the range of 90.0 to 107.0%. The method offers high separation efficiency, short analysis time, small sample consumption, and good repeatability.
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Simultaneous determination of dihydroxybenzene and phenylenediamine positional isomers using capillary zone electrophoresis coupled with amperometric detection
Journal of separation science, 2009Co-Authors: Shuqing Dong, Qingjiang Wang, Langzhu Chi, Zhiyong Yang, Yuzhi FangAbstract:In general capillary zone electrophoresis (CZE) separation models, o-, m-, and p-phenylenediamine isomers can be separated in a weak acidic Running Buffer for their pK(a) values being 4.52, 5.64, 6.04, respectively, while o-, m-, and p-dihydroxybenzene isomers can be separated in a weak basic Buffer for their pK(a) values being 9.40, 9.40 and 10.04, respectively. So, it is hard to find a suitable Running Buffer at a fixed pH in normal CZE for simultaneous separation of these two groups of positional isomers. In this paper, a novel method based on alternately Running two different pH Buffers in CZE coupled with amperometric detection (CZE-AD) was designed to simultaneously determine these two groups of positional isomers. It is found that when two different pH Running Buffers were employed alternately under appropriate order and time, the six analytes could be separated perfectly in less than 20 min and the detection limits were as low as 10(-7) mol/L. Furthermore, the effects of working electrode potential, pH and concentration of Running Buffer, separation voltage and injection time on CZE-AD were investigated. Experimental results demonstrated that the introduced method was practical to simultaneously determine two groups of positional isomers with different pK(a) and had some advantages of high sensitivity, good repeatability and small sample requirement.
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Simultaneous determination of flavonoids in chrysanthemum by capillary zone electrophoresis with Running Buffer modifiers
Talanta, 2008Co-Authors: Shan Zhang, Qingjiang Wang, Shuqing Dong, Langzhu Chi, Yuzhi FangAbstract:Abstract Despite the separation efficiency of capillary electrophoresis (CE) is much higher than other chromatographic methods, it is sometimes difficult to perfectly separate the complex ingredients in biological samples. One possible and simple way to develop the separation effect in CE is to add some modifiers in the Running Buffer. In this paper, the suitable Running Buffer modifiers were explored to simultaneously separate and detect six typical flavonoids (apigenin, luteolin, kaempferol, quercetin, (+)-catechin and (−)-epicatechin) which are the main active ingredients in chrysanthemum by capillary zone electrophoresis with amperometric detection (CZE-AD). It was found that when β-cyclodextrin (β-CD) and the mixture of methanol and ethanol were used as Running Buffer modifiers, a baseline separation of the six analytes could be accomplished in less than 20 min and the detection limits were as low as 10−7 or 10−8 g ml−1. Other factors affecting the CZE separation, such as working potential, pH value and ionic strength of Running Buffer, separation voltage and sample injection time were extensively investigated. Under the optimum conditions, a successful practical application on the determination of chrysanthemum samples confirmed the validity and practicability of this method.
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simultaneous determination of active ingredients in ethnomedicine gaultheria leucocarpa var yunnanensis and its medicinal preparation by capillary electrophoresis with electrochemical detection
Journal of Chromatographic Science, 2007Co-Authors: Yikun Zeng, Qingjiang Wang, Zhuxing Tang, Yuzhi FangAbstract:A simple and rapid capillary electrophoresis (CE) with electrcochemical detection (ED) method has been established for the simultaneous determination of seven active ingredients in the stems and roots of Gaultheria leucocarpa var. yunnanensis and its medicinal preparation, including (+)-catechin, rutin, gentisic acid, vallinic acid, salicylic acid, quercetin, and protocatechuic acid. The effects of working potential, pH, and concentration of Running Buffer, separation voltage, and injection time on CE‐ED are systematically investigated. Under the optimum conditions, the seven analytes could be completely separated within 23 min in a borax Running Buffer (pH 8.7). A good linear relationship is obtained over three orders of magnitude with detection limits (signal-to-noise ratio = 3) ranging from 5 ◊ 10 ‐8 g/mL to 3 ◊ 10‐7 g/mL for the analytes. The proposed method is successfully used in the analysis of real samples after a relatively simple extraction procedure, and the assay results are satisfactory.
Taufika Islam Williams - One of the best experts on this subject based on the ideXlab platform.
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A novel Bicine Running Buffer system for doubled sodium dodecyl sulfate – polyacrylamide gel electrophoresis of membrane proteins
Electrophoresis, 2006Co-Authors: Taufika Islam Williams, Jennifer C. Combs, Anup P. Thakur, Herbert J. Strobel, Bert C. LynnAbstract:A novel, Bicine-based SDS-PAGE Buffer system was developed for the analysis of membrane proteins. The method involves molecular weight-based separations of fully denatured and solubilized proteins in two dimensions. This doubled SDS-PAGE (dSDS-PAGE) approach produced a diagonal arrangement of protein spots and successfully circumvented problems associated with membrane proteome analysis involving traditional gel-based methods. Membrane proteins from the anaerobic bacterium Clostridium thermocellum were used for these investigations. Tricine-dSDS-PAGE and the newly developed Bicine-dSDS-PAGE were compared with the standard glycine-dSDS-PAGE (Laemmli protocol) in their suitability to separate C. thermocellum membrane proteins. Large-format gel experiments using optimized gel preparation and Running Buffer conditions revealed a 112% increase in protein spot count for Tricine-dSDS-PAGE and a 151% increase for Bicine-dSDS-PAGE, compared to glycine-dSDS-PAGE. The data clearly indicated that Bicine-dSDS-PAGE is a superior method for the analysis of membrane proteins, providing enhanced resolution and protein representation.
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a novel bicine Running Buffer system for doubled sodium dodecyl sulfate polyacrylamide gel electrophoresis of membrane proteins
Electrophoresis, 2006Co-Authors: Taufika Islam Williams, Jennifer C. Combs, Anup P. Thakur, Herbert J. Strobel, Bert C. LynnAbstract:A novel, Bicine-based SDS-PAGE Buffer system was developed for the analysis of membrane proteins. The method involves molecular weight-based separations of fully denatured and solubilized proteins in two dimensions. This doubled SDS-PAGE (dSDS-PAGE) approach produced a diagonal arrangement of protein spots and successfully circumvented problems associated with membrane proteome analysis involving traditional gel-based methods. Membrane proteins from the anaerobic bacterium Clostridium thermocellum were used for these investigations. Tricine-dSDS-PAGE and the newly developed Bicine-dSDS-PAGE were compared with the standard glycine-dSDS-PAGE (Laemmli protocol) in their suitability to separate C. thermocellum membrane proteins. Large-format gel experiments using optimized gel preparation and Running Buffer conditions revealed a 112% increase in protein spot count for Tricine-dSDS-PAGE and a 151% increase for Bicine-dSDS-PAGE, compared to glycine-dSDS-PAGE. The data clearly indicated that Bicine-dSDS-PAGE is a superior method for the analysis of membrane proteins, providing enhanced resolution and protein representation.
Shuqing Dong - One of the best experts on this subject based on the ideXlab platform.
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Simultaneous determination of phenylethanoid glycosides and aglycones by capillary zone electrophoresis with Running Buffer modifier
Analytical biochemistry, 2013Co-Authors: Shuqing Dong, Ruibin Gao, Yan Yang, Mei Guo, Liang ZhaoAbstract:Although the separation efficiency of capillary electrophoresis (CE) is much higher than that of other chromatographic methods, it is sometimes difficult to adequately separate the complex ingredients in biological samples. This article describes how one effective and simple way to develop the separation efficiency in CE is to add some modifiers to the Running Buffer. The suitable Running Buffer modifier β-cyclodextrin (β-CD) was explored to fast and completely separate four phenylethanoid glycosides and aglycones (homovanillyl alcohol, hydroxytyrosol, 3,4-dimethoxycinnamic acid, and caffeic acid) in Lamiophlomis rotata (Lr) and Cistanche by capillary zone electrophoresis with ultraviolet (UV) detection. It was found that when β-CD was used as Running Buffer modifier, a baseline separation of the four analytes could be accomplished in less than 20 min and the detection limits were as low as 10(-3) mg L(-1). Other factors affecting the CE separation, such as working potential, pH value and ionic strength of Running Buffer, separation voltage, and sample injection time, were investigated extensively. Under the optimal conditions, a successful practical application on the determination of Lr and Cistanche samples confirmed the validity and practicability of this method.
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Simultaneous determination of flavonoids and phenolic acids in Chinese herbal tea by beta-cyclodextrin based capillary zone electrophoresis
Mikrochimica Acta, 2009Co-Authors: Langzhu Chi, Shuqing Dong, Qingjiang Wang, Yuzhi FangAbstract:Kaempferol, apigenin, rutin, quercetin, luteolin and ferulic acid are separated and detected in Chinese herbal tea using capillary zone electrophoresis coupled to amperometric detection. The phosphate Running Buffer also contains s-cyclodextrin (s-CD), which assists in separation and gives excellent separations within 20 min and detection limits as low as 10 ng mL−1 (S/N = 3). The effects of working electrode potential, pH and concentration of Running Buffer, concentration of s-CD, separation voltage and injection time were investigated. The method was applied to analyze tea samples with recoveries in the range of 90.0 to 107.0%. The method offers high separation efficiency, short analysis time, small sample consumption, and good repeatability.
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Simultaneous determination of dihydroxybenzene and phenylenediamine positional isomers using capillary zone electrophoresis coupled with amperometric detection
Journal of separation science, 2009Co-Authors: Shuqing Dong, Qingjiang Wang, Langzhu Chi, Zhiyong Yang, Yuzhi FangAbstract:In general capillary zone electrophoresis (CZE) separation models, o-, m-, and p-phenylenediamine isomers can be separated in a weak acidic Running Buffer for their pK(a) values being 4.52, 5.64, 6.04, respectively, while o-, m-, and p-dihydroxybenzene isomers can be separated in a weak basic Buffer for their pK(a) values being 9.40, 9.40 and 10.04, respectively. So, it is hard to find a suitable Running Buffer at a fixed pH in normal CZE for simultaneous separation of these two groups of positional isomers. In this paper, a novel method based on alternately Running two different pH Buffers in CZE coupled with amperometric detection (CZE-AD) was designed to simultaneously determine these two groups of positional isomers. It is found that when two different pH Running Buffers were employed alternately under appropriate order and time, the six analytes could be separated perfectly in less than 20 min and the detection limits were as low as 10(-7) mol/L. Furthermore, the effects of working electrode potential, pH and concentration of Running Buffer, separation voltage and injection time on CZE-AD were investigated. Experimental results demonstrated that the introduced method was practical to simultaneously determine two groups of positional isomers with different pK(a) and had some advantages of high sensitivity, good repeatability and small sample requirement.
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Simultaneous determination of flavonoids in chrysanthemum by capillary zone electrophoresis with Running Buffer modifiers
Talanta, 2008Co-Authors: Shan Zhang, Qingjiang Wang, Shuqing Dong, Langzhu Chi, Yuzhi FangAbstract:Abstract Despite the separation efficiency of capillary electrophoresis (CE) is much higher than other chromatographic methods, it is sometimes difficult to perfectly separate the complex ingredients in biological samples. One possible and simple way to develop the separation effect in CE is to add some modifiers in the Running Buffer. In this paper, the suitable Running Buffer modifiers were explored to simultaneously separate and detect six typical flavonoids (apigenin, luteolin, kaempferol, quercetin, (+)-catechin and (−)-epicatechin) which are the main active ingredients in chrysanthemum by capillary zone electrophoresis with amperometric detection (CZE-AD). It was found that when β-cyclodextrin (β-CD) and the mixture of methanol and ethanol were used as Running Buffer modifiers, a baseline separation of the six analytes could be accomplished in less than 20 min and the detection limits were as low as 10−7 or 10−8 g ml−1. Other factors affecting the CZE separation, such as working potential, pH value and ionic strength of Running Buffer, separation voltage and sample injection time were extensively investigated. Under the optimum conditions, a successful practical application on the determination of chrysanthemum samples confirmed the validity and practicability of this method.
