The Experts below are selected from a list of 249 Experts worldwide ranked by ideXlab platform
Pier Giorgio Righetti - One of the best experts on this subject based on the ideXlab platform.
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steady state electrolysis of an Ampholyte solution and possibility of violation of the law of ph monotony
Electrophoresis, 1998Co-Authors: Alexander V Stoyanov, Pier Giorgio RighettiAbstract:The problem of stationary electrolysis of a single-component Ampholyte solution is analyzed. The effect of nonconstant relative mobility is taken into account. It is demonstrated that an incorrect dissociation model (i.e., expressing the resulting Ampholyte flux by the arithmetic sum of the fluxes for "cationic" and "anionic" species) may lead to the violation of the so-called "law of pH monotony".
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Ampholyte dissociation theory and properties of Ampholyte aqueous solutions
Electrophoresis, 1997Co-Authors: Alexander V Stoyanov, Pier Giorgio RighettiAbstract:Two different approaches (stepwise and parallel mechanism) for describing the dissociation of amphoteric protolytes are described. Additionally, the classification of the possible model on the basis of the Ampholyte lifetime state is proposed. The possibility of application of different schemes is discussed. It is suggested that the correct description may be based only on highly relaxing models. The formulas describing the properties of Ampholyte solutions are derived for the case of two ionogenic groups. It is demonstrated that the incorrect choice of the dissociation model may result in essential mistakes in calculation of such values as buffer capacity, electrophoretic mobility, and conductivity. On the basis of the theory here developed, the interpretation of natural pH-gradient conductivity is given. Some terminology questions are also discussed.
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capillary zone electrophoresis of oligonucleotides in isoelectric buffers and against a stationary ph gradient
Electrophoresis, 1997Co-Authors: Alexander V Stoyanov, Cecilia Gelfi, Pier Giorgio RighettiAbstract:Capillary zone electrophoresis of oligonucleotides in a background electrolyte of two different types of stationary buffers is proposed: single, isoelectric amphoteres and focused carrier Ampholytes. In the first case, two zwitterionic molecules are evaluated: lysine and histidine. Although the former has a five times higher buffering power (beta) at the pI (9.74) than the latter (pI 7.47), due to the favorable delta pK value (1.6 vs. 3) and thus should be the preferred species, a new parameter for evaluating the performance of isoelectric buffers is proposed: the beta/lambda ratio, i.e., the ratio between the buffering power and its conductivity. Ideal buffers are those with the highest beta/lambda ratio, since this allows delivering very high voltage gradients with minimal Joule effects. Since the pI of Lys is situated in a pH region (9.74) where bulk water begins to conduct, whereas His has a pI close to neutrality, the beta/lambda ratio is more favorable for His than for Lys. In the second case (zone electrophoresis of oligonucleotides against a preformed pH gradient), it is shown that migration against a pH 6.5-10 Pharmalyte carrier Ampholyte pH gradient offers a unique analyte resolution. This is possibly due to two effects: (i) When injected at the alkaline extreme (ca. pH 10) of the pH gradient, the oligonucleotide zones undergo a stacking effect, with consequent zone sharpening, due to modulation of their free mobility via protonation of the -OH group (enolate ion) in the hetero aromatic rings of G and T, which undergo a lactam-lactim transition. (ii) As the zones migrate down the pH gradient, they transit through a pH 6.5-8.5 zone where, for Pharmalytes, the beta/lambda ratio reaches a maximum and is constant as well. This last condition allows high voltage gradients (typically 1000 V/cm, even in 75 microm capillaries) to be delivered, thus greatly reducing the analysis time and maintaining peak sharpness, due to limited diffusion.
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screening of umbilical cord blood hemoglobins by isoelectric focusing in capillaries
Electrophoresis, 1995Co-Authors: Monica Conti, Cecilia Gelfi, Pier Giorgio RighettiAbstract:Separation and quantitation of the three main hemoglobin components of umbilical cord blood (fetal, acetylated fetal and adult hemoglobins; Hb F, Fac, A) by capillary isoelectric focusing (IEF) in a pH 6-8 gradient is reported. Even in coated capillaries (with covalently bound chains of linear acrylamido derivatives, notably N-acryloylaminoethoxyethanol), no base line separation is obtained between Hb F and A, although this is routinely achieved in gel slab IEF. However, when the carrier Ampholyte buffers were added to 3% short-chain liquid linear polyacrylamide, base line resolution and stabilization of peak transit times were obtained. This suggests that even in the best coating procedures, patches of the inner capillary surface could still be naked, so that the static coating is complemented by a dynamic coating on the unoccupied sites. An additional improvement in separation occurs if the above mixture, comprising 5% carrier Ampholytes in the pH 6-8 range and 3% soluble polyacrylamide, is made to contain 50 mM beta-alanine, a "separator" known to flatten the pH gradient around pH 7. In the normal newborns analyzed (n = 30), the following average values were obtained: Hb F, 70.1% (range 65-75%); Hb A, 20.2% (range 15-25%); and HbFac, 9.5% (range 7-11%).
Prashanta Dutta - One of the best experts on this subject based on the ideXlab platform.
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steady state protein focusing in carrier Ampholyte based isoelectric focusing part ii validation and case studies
Electrophoresis, 2017Co-Authors: Jaesool Shim, Kisoo Yoo, Prashanta DuttaAbstract:In this study, we systematically investigate the validity and applicability of an analytical model developed for carrier Ampholyte-based isoelectric focusing (IEF). Three different IEF cases are considered in order to evaluate the efficacy of the approximate analytical results by comparison with high-resolution computer simulations. In the first case, three proteins are separated in a narrow pH range (6-9) using 50 carrier Ampholytes. In the second and third cases, the separation of proteins is studied in broad pH range (3-10) IEF using 100 carrier Ampholytes. Results obtained from the approximate analytical models are in very good agreement with the numerical results for IEF separation of cardiac troponin I, albumin, and hemoglobin in both narrow and broad pH ranges. The sensitivity of the analytical model is also tested for different initial mass ratios of proteins to Ampholytes. No appreciable differences are observed between approximate analytical and numerical results within the mass ratio range studied. The effect of a nominal electric field and/or a nominal pH gradient on protein focusing is also examined to demonstrate the effectiveness of the analytical model. Our results indicate that the use of both nominal electric field and pH gradient will result in erroneous peak concentrations for proteins. Finally, we describe the limitations of the approximate analytical solutions. This article is protected by copyright. All rights reserved
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steady state protein focusing in carrier Ampholyte based isoelectric focusing part i analytical solution
Electrophoresis, 2017Co-Authors: Jaesool Shim, Kisoo Yoo, Prashanta DuttaAbstract:The determination of an analytical solution to find the steady state protein concentration distribution in isoelectric focusing (IEF) is very challenging due to the nonlinear coupling between mass and charge conservation equations. In this study, approximate analytical solutions are obtained for steady state protein distribution in carrier Ampholyte-based IEF. Similar to the work of Svensson, the final concentration profile for proteins is assumed to be Gaussian, but appropriate expressions are presented in order to obtain the effective electric field and pH gradient in the focused protein band region. Analytical results are found from iterative solutions of a system of coupled algebraic equations using only several iterations for IEF separation of three plasma proteins: albumin, cardiac troponin I (cTnI), and hemoglobin. The analytical results are compared with numerically predicted results for IEF, showing excellent agreement. Analytically obtained electric field and ionic conductivity distributions show significant deviation from their nominal values, which is essential in finding the protein focusing behavior at isoelectric points. These analytical solutions can be used to determine steady-state protein concentration distribution for experiment design of IEF considering any number of proteins and Ampholytes. Moreover, the model presented herein can be used to find the conductivity, electric field, and pH field in the separation channel. This article is protected by copyright. All rights reserved
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effects of Ampholyte dissociation constants on protein separation in on chip isoelectric focusing
Journal of Nanoscience and Nanotechnology, 2008Co-Authors: Jaesool Shim, Prashanta Dutta, Cornelius F IvoryAbstract:Numerical simulations are presented for Ampholyte-based isoelectric focusing in 2D microgeometries. In this study, model proteins are focused in the presence of 25 biprotic Ampholytes under an applied electric field. Each protein is considered as a simple polypeptide having ten charge states, while the biprotic Ampholytes are selected to generate a shallow pH range of 6 to 9. Straight and contraction-expansion microchannels are considered here, and a nominal electric field of 300 V/cm is maintained for separation of proteins. Six distinct values of deltapKs between 1 and 3.5 are investigated for Ampholytes to form pH profiles in a 1 cm long microchannel. Simulation results show that relatively larger values of deltapK(deltapK > 3) are required to form stepless pH profiles in the system. The peak heights and differential resolutions of focused proteins are much higher for lower values of deltapK for which a stepped pH profile is evident. For each protein, the time it takes for the two edges of a peak to merge increases linearly with deltapK, while the focusing time goes up exponentially with increasing deltapK. Both merging and focusing times of protein are higher for contraction-expansion microchannel than those of straight microchannel. For a particular value of deltapK, the contracted "Zoom" region of contraction-expansion channel is able to form more tightly focused bands than the expanded region.
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effects of Ampholyte concentration on protein behavior in on chip isoelectric focusing
Electrophoresis, 2008Co-Authors: Jaesool Shim, Prashanta Dutta, Cornelius F IvoryAbstract:The effects of mobility corrections on carrier Ampholytes are studied at various Ampholyte concentrations to understand protein behavior during IEF. IEF simulations are conducted in the presence of 25 biprotic carrier Ampholytes within a pH range of 6-9 after applying the Onsager-Debye-Huckel correction to the carrier Ampholytes. Two model proteins with ten charge states but without ionic strength corrections are allowed to focus under an electric field of 300 V/cm in a 1 cm long channel. The IEF simulation results show that higher ionic strengths (50 - 100 mM) cause significant changes in the transient movement as well as the final focused profiles of both Ampholytes and proteins. The time required for a single, well-defined peak to form increases with ionic strength when Onsager corrections are applied to the carrier Ampholytes. For a particular Ampholyte concentration, the space-averaged conductivity does not change during the final focusing stage, but the magnitude of space averaged conductivity is different for different Ampholyte concentration. The simulation results also reveal that at steady-state ionic strength profiles remain flat throughout the channel except at the locations of proteins where a significant change in Ampholyte concentration is obtained.
Alexander V Stoyanov - One of the best experts on this subject based on the ideXlab platform.
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conductivity properties of carrier Ampholyte ph gradients in isoelectric focusing
Electrophoresis, 2005Co-Authors: Alexander V Stoyanov, Champak Das, Carl K Fredrickson, Hugh Z FanAbstract:The conductivity properties of natural pH gradient created by carrier Ampholytes were studied during the process of isoelectric focusing (IEF). IEF was performed in capillaries (10-30 mm long) or in microchips with the same channel length. A 10-30x reduction of the conductivity of the separation medium was observed during the establishment of pH gradient. Results obtained using different IEF voltages indicate that there is a nonlinear relationship between the conductivity of an established pH gradient and the applied electric field. Our theoretical analysis using a simplified model generated values that reasonably agree with the experimental data. In addition, we found that above a certain electric field ( approximately 300 V/cm), resolution does not increase with the applied voltage as predicated; we observed band-broadening and gel breakdown. The approach presented in this work can be used for optimization of the IEF separation and judicious selection of IEF conditions.
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steady state electrolysis of an Ampholyte solution and possibility of violation of the law of ph monotony
Electrophoresis, 1998Co-Authors: Alexander V Stoyanov, Pier Giorgio RighettiAbstract:The problem of stationary electrolysis of a single-component Ampholyte solution is analyzed. The effect of nonconstant relative mobility is taken into account. It is demonstrated that an incorrect dissociation model (i.e., expressing the resulting Ampholyte flux by the arithmetic sum of the fluxes for "cationic" and "anionic" species) may lead to the violation of the so-called "law of pH monotony".
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Ampholyte dissociation theory and properties of Ampholyte aqueous solutions
Electrophoresis, 1997Co-Authors: Alexander V Stoyanov, Pier Giorgio RighettiAbstract:Two different approaches (stepwise and parallel mechanism) for describing the dissociation of amphoteric protolytes are described. Additionally, the classification of the possible model on the basis of the Ampholyte lifetime state is proposed. The possibility of application of different schemes is discussed. It is suggested that the correct description may be based only on highly relaxing models. The formulas describing the properties of Ampholyte solutions are derived for the case of two ionogenic groups. It is demonstrated that the incorrect choice of the dissociation model may result in essential mistakes in calculation of such values as buffer capacity, electrophoretic mobility, and conductivity. On the basis of the theory here developed, the interpretation of natural pH-gradient conductivity is given. Some terminology questions are also discussed.
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capillary zone electrophoresis of oligonucleotides in isoelectric buffers and against a stationary ph gradient
Electrophoresis, 1997Co-Authors: Alexander V Stoyanov, Cecilia Gelfi, Pier Giorgio RighettiAbstract:Capillary zone electrophoresis of oligonucleotides in a background electrolyte of two different types of stationary buffers is proposed: single, isoelectric amphoteres and focused carrier Ampholytes. In the first case, two zwitterionic molecules are evaluated: lysine and histidine. Although the former has a five times higher buffering power (beta) at the pI (9.74) than the latter (pI 7.47), due to the favorable delta pK value (1.6 vs. 3) and thus should be the preferred species, a new parameter for evaluating the performance of isoelectric buffers is proposed: the beta/lambda ratio, i.e., the ratio between the buffering power and its conductivity. Ideal buffers are those with the highest beta/lambda ratio, since this allows delivering very high voltage gradients with minimal Joule effects. Since the pI of Lys is situated in a pH region (9.74) where bulk water begins to conduct, whereas His has a pI close to neutrality, the beta/lambda ratio is more favorable for His than for Lys. In the second case (zone electrophoresis of oligonucleotides against a preformed pH gradient), it is shown that migration against a pH 6.5-10 Pharmalyte carrier Ampholyte pH gradient offers a unique analyte resolution. This is possibly due to two effects: (i) When injected at the alkaline extreme (ca. pH 10) of the pH gradient, the oligonucleotide zones undergo a stacking effect, with consequent zone sharpening, due to modulation of their free mobility via protonation of the -OH group (enolate ion) in the hetero aromatic rings of G and T, which undergo a lactam-lactim transition. (ii) As the zones migrate down the pH gradient, they transit through a pH 6.5-8.5 zone where, for Pharmalytes, the beta/lambda ratio reaches a maximum and is constant as well. This last condition allows high voltage gradients (typically 1000 V/cm, even in 75 microm capillaries) to be delivered, thus greatly reducing the analysis time and maintaining peak sharpness, due to limited diffusion.
Jaesool Shim - One of the best experts on this subject based on the ideXlab platform.
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steady state protein focusing in carrier Ampholyte based isoelectric focusing part ii validation and case studies
Electrophoresis, 2017Co-Authors: Jaesool Shim, Kisoo Yoo, Prashanta DuttaAbstract:In this study, we systematically investigate the validity and applicability of an analytical model developed for carrier Ampholyte-based isoelectric focusing (IEF). Three different IEF cases are considered in order to evaluate the efficacy of the approximate analytical results by comparison with high-resolution computer simulations. In the first case, three proteins are separated in a narrow pH range (6-9) using 50 carrier Ampholytes. In the second and third cases, the separation of proteins is studied in broad pH range (3-10) IEF using 100 carrier Ampholytes. Results obtained from the approximate analytical models are in very good agreement with the numerical results for IEF separation of cardiac troponin I, albumin, and hemoglobin in both narrow and broad pH ranges. The sensitivity of the analytical model is also tested for different initial mass ratios of proteins to Ampholytes. No appreciable differences are observed between approximate analytical and numerical results within the mass ratio range studied. The effect of a nominal electric field and/or a nominal pH gradient on protein focusing is also examined to demonstrate the effectiveness of the analytical model. Our results indicate that the use of both nominal electric field and pH gradient will result in erroneous peak concentrations for proteins. Finally, we describe the limitations of the approximate analytical solutions. This article is protected by copyright. All rights reserved
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steady state protein focusing in carrier Ampholyte based isoelectric focusing part i analytical solution
Electrophoresis, 2017Co-Authors: Jaesool Shim, Kisoo Yoo, Prashanta DuttaAbstract:The determination of an analytical solution to find the steady state protein concentration distribution in isoelectric focusing (IEF) is very challenging due to the nonlinear coupling between mass and charge conservation equations. In this study, approximate analytical solutions are obtained for steady state protein distribution in carrier Ampholyte-based IEF. Similar to the work of Svensson, the final concentration profile for proteins is assumed to be Gaussian, but appropriate expressions are presented in order to obtain the effective electric field and pH gradient in the focused protein band region. Analytical results are found from iterative solutions of a system of coupled algebraic equations using only several iterations for IEF separation of three plasma proteins: albumin, cardiac troponin I (cTnI), and hemoglobin. The analytical results are compared with numerically predicted results for IEF, showing excellent agreement. Analytically obtained electric field and ionic conductivity distributions show significant deviation from their nominal values, which is essential in finding the protein focusing behavior at isoelectric points. These analytical solutions can be used to determine steady-state protein concentration distribution for experiment design of IEF considering any number of proteins and Ampholytes. Moreover, the model presented herein can be used to find the conductivity, electric field, and pH field in the separation channel. This article is protected by copyright. All rights reserved
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effects of Ampholyte dissociation constants on protein separation in on chip isoelectric focusing
Journal of Nanoscience and Nanotechnology, 2008Co-Authors: Jaesool Shim, Prashanta Dutta, Cornelius F IvoryAbstract:Numerical simulations are presented for Ampholyte-based isoelectric focusing in 2D microgeometries. In this study, model proteins are focused in the presence of 25 biprotic Ampholytes under an applied electric field. Each protein is considered as a simple polypeptide having ten charge states, while the biprotic Ampholytes are selected to generate a shallow pH range of 6 to 9. Straight and contraction-expansion microchannels are considered here, and a nominal electric field of 300 V/cm is maintained for separation of proteins. Six distinct values of deltapKs between 1 and 3.5 are investigated for Ampholytes to form pH profiles in a 1 cm long microchannel. Simulation results show that relatively larger values of deltapK(deltapK > 3) are required to form stepless pH profiles in the system. The peak heights and differential resolutions of focused proteins are much higher for lower values of deltapK for which a stepped pH profile is evident. For each protein, the time it takes for the two edges of a peak to merge increases linearly with deltapK, while the focusing time goes up exponentially with increasing deltapK. Both merging and focusing times of protein are higher for contraction-expansion microchannel than those of straight microchannel. For a particular value of deltapK, the contracted "Zoom" region of contraction-expansion channel is able to form more tightly focused bands than the expanded region.
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effects of Ampholyte concentration on protein behavior in on chip isoelectric focusing
Electrophoresis, 2008Co-Authors: Jaesool Shim, Prashanta Dutta, Cornelius F IvoryAbstract:The effects of mobility corrections on carrier Ampholytes are studied at various Ampholyte concentrations to understand protein behavior during IEF. IEF simulations are conducted in the presence of 25 biprotic carrier Ampholytes within a pH range of 6-9 after applying the Onsager-Debye-Huckel correction to the carrier Ampholytes. Two model proteins with ten charge states but without ionic strength corrections are allowed to focus under an electric field of 300 V/cm in a 1 cm long channel. The IEF simulation results show that higher ionic strengths (50 - 100 mM) cause significant changes in the transient movement as well as the final focused profiles of both Ampholytes and proteins. The time required for a single, well-defined peak to form increases with ionic strength when Onsager corrections are applied to the carrier Ampholytes. For a particular Ampholyte concentration, the space-averaged conductivity does not change during the final focusing stage, but the magnitude of space averaged conductivity is different for different Ampholyte concentration. The simulation results also reveal that at steady-state ionic strength profiles remain flat throughout the channel except at the locations of proteins where a significant change in Ampholyte concentration is obtained.
Matthew I. Gibson - One of the best experts on this subject based on the ideXlab platform.
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synthetically scalable poly Ampholyte which dramatically enhances cellular cryopreservation
Biomacromolecules, 2019Co-Authors: Trisha L Bailey, Christopher D Stubbs, Kathryn Murray, Ruben M F Tomas, Lucienne Otten, Matthew I. GibsonAbstract:The storage and transport of frozen cells underpin the emerging/existing cell-based therapies and are used in every biomedical research lab globally. The current gold-standard cryoprotectant dimethyl sulfoxide (DMSO) does not give quantitative cell recovery in suspension or in two-dimensional (2D) or three-dimensional (3D) cell models, and the solvent and cell debris must be removed prior to application/transfusion. There is a real need to improve this 50-year-old method to underpin emerging regenerative and cell-based therapies. Here, we introduce a potent and synthetically scalable polymeric cryopreservation enhancer which is easily obtained in a single step from a low cost and biocompatible precursor, poly(methyl vinyl ether-alt-maleic anhydride). This poly(Ampholyte) enables post-thaw recoveries of up to 88% for a 2D cell monolayer model compared to just 24% using conventional DMSO cryopreservation. The poly(Ampholyte) also enables reduction of [DMSO] from 10 wt % to just 2.5 wt % in suspension cryopreservation, which can reduce the negative side effects and speed up post-thaw processing. After thawing, the cells have reduced membrane damage and faster growth rates compared to those without the polymer. The polymer appears to function by a unique extracellular mechanism by stabilization of the cell membrane, rather than by modulation of ice formation and growth. This new macromolecular cryoprotectant will find applications across basic and translational biomedical science and may improve the cold chain for cell-based therapies.
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rational yet simple design and synthesis of an antifreeze protein inspired polymer for cellular cryopreservation
Chemical Communications, 2015Co-Authors: Daniel E Mitchell, Neil R Cameron, Matthew I. GibsonAbstract:Antifreeze (glyco) proteins AF(G)Ps are potent ice recrystallization inhibitors, which is a desirable property to enhance cryopreservation of donor tissue/cells. Here we present the rational synthesis of a new, biomimetic, ice-recrystallization inhibiting polymer derived from a cheap commodity polymer, based on an Ampholyte structure. The polymer is used to enhance the cryopreservation of red blood cells, demonstrating a macromolecular solution to tissue storage.
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quantitative study on the antifreeze protein mimetic ice growth inhibition properties of poly Ampholytes derived from vinyl based polymers
Biomaterials Science, 2014Co-Authors: Daniel E Mitchell, Mary Lilliman, Sebastian G Spain, Matthew I. GibsonAbstract:Antifreeze (glyco) proteins (AF(G)Ps) from the blood of polar fish species are extremely potent ice recrystallization inhibitors (IRI), but are difficult to synthesise or extract from natural sources. Despite this challenge, materials which display IRI are appealing due to their ability to enhance cellular cryopreservation, for applications including regenerative and transplantation medicine. Here, poly(Ampholytes), which contain a mixture of cationic and anionic side chains are quantitatively evaluated for their IRI activity. Poly(aminoethyl methacrylate), obtained by RAFT polymerization, is functionalised with succinic anhydride to generate the poly(Ampholytes). The charge balance of the side chains is shown to be crucial, with only 50 : 50 mixtures having strong IRI activity, which also scales with molecular weight. This is the first example of a non-hydroxylated synthetic polymer with quantifiable IRI activity and raises questions about the mechanism of IRI, as the polymers have no obvious ice-binding motif. The ampholytic structure is shown to be transferable to carbohydrate-centred polymers with activity retained, but poly(betaines) are shown to be inactive.