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Sergey N Krylov - One of the best experts on this subject based on the ideXlab platform.

  • empirical predictor of conditions that support Ideal Filter capillary electrophoresis
    Electrophoresis, 2020
    Co-Authors: Tong Ye Wang, Liang Hu, Sergey N Krylov
    Abstract:

    : 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.

  • Ideal Filter capillary electrophoresis a highly efficient partitioning method for selection of protein binders from oligonucleotide libraries
    Electrophoresis, 2019
    Co-Authors: An T. H. Le, Svetlana M. Krylova, Sergey N Krylov
    Abstract:

    : Selection of affinity ligands for protein targets from oligonucleotide libraries currently involves multiple rounds of alternating steps of partitioning of protein-bound oligonucleotides (binders) from protein-unbound oligonucleotides (nonbinders). We have recently introduced Ideal-Filter capillary electrophoresis (IFCE) for binder selection in a single step of partitioning. In IFCE, protein-binder complexes and nonbinders move inside the capillary in the opposite directions, and the efficiency of their partitioning reaches 109 , i.e., only one of a billion molecules of nonbinders leaks through IFCE while all binders pass through. The condition of IFCE can be satisfied when the magnitude of the mobility of EOF is smaller than that of the protein-binder complexes and larger than that of nonbinders. The efficiency of partitioning in IFCE is 10 million times higher than those of solid-phase-based methods of partitioning typically used in selection of affinity ligands for protein targets from oligonucleotide libraries. Here, we provide additional details on our justification for IFCE development. We elaborate on electrophoretic aspects of the method and define the theoretical range of EOF mobilities that support IFCE. Based on these theoretical results, we identify an experimental range of background electrolyte's ionic strength that supports IFCE. We also extend our interpretation of the results and discuss in-depth IFCE's prospective in practical applications and fundamental studies.

  • determination of the equilibrium constant and rate constant of protein oligonucleotide complex dissociation under the conditions of Ideal Filter capillary electrophoresis
    Analytical Chemistry, 2019
    Co-Authors: An T. H. Le, Svetlana M. Krylova, Sergey N Krylov
    Abstract:

    Ideal-Filter capillary electrophoresis (IFCE) allows selection of protein binders from oligonucleotide libraries in a single step of partitioning in which protein-bound and unbound oligonucleotides move in the opposite directions. In IFCE, the unbound oligonucleotide does not reach the detector, imposing a problem for finding the equilibrium constant (Kd) and rate constant (koff) of protein–oligonucleotide complex dissociation. We report a double-passage approach that allows finding Kd and koff under the IFCE conditions, i.e. near-physiological pH and ionic strength. First, a plug of the protein–oligonucleotide equilibrium mixture passes to the detector in a pressure-driven flow, allowing for both the complex and free oligonucleotide to be detected as a single first peak. Second, the pressure is turned off and the voltage is applied to reverse the migration of only the complex which is detected as the second peak. The experiment is repeated with a lower voltage consequently resulting in longer travel time...

  • Ideal Filter capillary electrophoresis ifce facilitates the one step selection of aptamers
    Angewandte Chemie, 2019
    Co-Authors: An T. H. Le, Svetlana M. Krylova, Mirzo Kanoatov, Shrey Desai, Sergey N Krylov
    Abstract:

    : Selection of aptamers from oligonucleotide libraries currently requires multiple rounds of alternating steps of partitioning of binders from nonbinders and enzymatic amplification of all collected oligonucleotides. Herein, we report a highly practical solution for reliable one-step selection of aptamers. We introduce partitioning by Ideal-Filter capillary electrophoresis (IFCE) in which binders and nonbinders move in the opposite directions. The efficiency of IFCE-based partitioning reaches 109 , which is ten million times higher than that of typical solid-phase partitioning methods. One step of IFCE-based partitioning is sufficient for the selection of a high-affinity aptamer pool for a protein target. Partitioning by IFCE promises to become an indispensable tool for fast and robust selection of binders from different types of oligonucleotide libraries.

An T. H. Le - One of the best experts on this subject based on the ideXlab platform.

  • Ideal Filter capillary electrophoresis a highly efficient partitioning method for selection of protein binders from oligonucleotide libraries
    Electrophoresis, 2019
    Co-Authors: An T. H. Le, Svetlana M. Krylova, Sergey N Krylov
    Abstract:

    : Selection of affinity ligands for protein targets from oligonucleotide libraries currently involves multiple rounds of alternating steps of partitioning of protein-bound oligonucleotides (binders) from protein-unbound oligonucleotides (nonbinders). We have recently introduced Ideal-Filter capillary electrophoresis (IFCE) for binder selection in a single step of partitioning. In IFCE, protein-binder complexes and nonbinders move inside the capillary in the opposite directions, and the efficiency of their partitioning reaches 109 , i.e., only one of a billion molecules of nonbinders leaks through IFCE while all binders pass through. The condition of IFCE can be satisfied when the magnitude of the mobility of EOF is smaller than that of the protein-binder complexes and larger than that of nonbinders. The efficiency of partitioning in IFCE is 10 million times higher than those of solid-phase-based methods of partitioning typically used in selection of affinity ligands for protein targets from oligonucleotide libraries. Here, we provide additional details on our justification for IFCE development. We elaborate on electrophoretic aspects of the method and define the theoretical range of EOF mobilities that support IFCE. Based on these theoretical results, we identify an experimental range of background electrolyte's ionic strength that supports IFCE. We also extend our interpretation of the results and discuss in-depth IFCE's prospective in practical applications and fundamental studies.

  • determination of the equilibrium constant and rate constant of protein oligonucleotide complex dissociation under the conditions of Ideal Filter capillary electrophoresis
    Analytical Chemistry, 2019
    Co-Authors: An T. H. Le, Svetlana M. Krylova, Sergey N Krylov
    Abstract:

    Ideal-Filter capillary electrophoresis (IFCE) allows selection of protein binders from oligonucleotide libraries in a single step of partitioning in which protein-bound and unbound oligonucleotides move in the opposite directions. In IFCE, the unbound oligonucleotide does not reach the detector, imposing a problem for finding the equilibrium constant (Kd) and rate constant (koff) of protein–oligonucleotide complex dissociation. We report a double-passage approach that allows finding Kd and koff under the IFCE conditions, i.e. near-physiological pH and ionic strength. First, a plug of the protein–oligonucleotide equilibrium mixture passes to the detector in a pressure-driven flow, allowing for both the complex and free oligonucleotide to be detected as a single first peak. Second, the pressure is turned off and the voltage is applied to reverse the migration of only the complex which is detected as the second peak. The experiment is repeated with a lower voltage consequently resulting in longer travel time...

  • Ideal Filter capillary electrophoresis ifce facilitates the one step selection of aptamers
    Angewandte Chemie, 2019
    Co-Authors: An T. H. Le, Svetlana M. Krylova, Mirzo Kanoatov, Shrey Desai, Sergey N Krylov
    Abstract:

    : Selection of aptamers from oligonucleotide libraries currently requires multiple rounds of alternating steps of partitioning of binders from nonbinders and enzymatic amplification of all collected oligonucleotides. Herein, we report a highly practical solution for reliable one-step selection of aptamers. We introduce partitioning by Ideal-Filter capillary electrophoresis (IFCE) in which binders and nonbinders move in the opposite directions. The efficiency of IFCE-based partitioning reaches 109 , which is ten million times higher than that of typical solid-phase partitioning methods. One step of IFCE-based partitioning is sufficient for the selection of a high-affinity aptamer pool for a protein target. Partitioning by IFCE promises to become an indispensable tool for fast and robust selection of binders from different types of oligonucleotide libraries.

  • Determination of the Equilibrium Constant and Rate Constant of Protein–Oligonucleotide Complex Dissociation under the Conditions of Ideal-Filter Capillary Electrophoresis
    Analytical Chemistry, 2019
    Co-Authors: An T. H. Le, Svetlana M. Krylova, Sergey N. Krylov And
    Abstract:

    Ideal-Filter capillary electrophoresis (IFCE) allows selection of protein binders from oligonucleotide libraries in a single step of partitioning in which protein-bound and unbound oligonucleotides move in the opposite directions. In IFCE, the unbound oligonucleotide does not reach the detector, imposing a problem for finding the equilibrium constant (K d) and rate constant (k off) of protein−oligonucleotide complex dissociation. We report a double-passage approach that allows finding K d and k off under the IFCE conditions, i.e. near-physiological pH and ionic strength. First, a plug of the protein−oligonucleotide equilibrium mixture passes to the detector in a pressure-driven flow, allowing for both the complex and free oligonucleotide to be detected as a single first peak. Second, the pressure is turned off and the voltage is applied to reverse the migration of only the complex which is detected as the second peak. The experiment is repeated with a lower voltage consequently resulting in longer travel time of the complex to the detector, greater extent of complex dissociation, and the decreased area of the second peak. Finally, the peak areas are used to calculate the values of K d and k off. Here we explain theoretical and practical aspects of the double-passage approach, prove its validity quantitatively, and, demonstrate its application to determine K d and k off for an affinity complex between a protein and its DNA aptamer. The double-passage approach for finding K d and k off of protein−oligonucleotide complexes under the IFCE conditions is a perfect complement for IFCE-based selection of protein binders from oligonucleotide libraries. S election of protein binders from oligonucleotide libraries, such as random-sequence oligonucleotide libraries and DNA-encoded libraries (DELs), can provide diverse pools of molecules for development of diagnostic probes and drugs. 1−7 Random-sequence oligonucleotide libraries are used to select aptamers, while DELs are used to select small-molecules, capable of binding target proteins. The low abundance of protein binders in oligonucleotide libraries makes it very hard to separate binders from nonbinders in a single step of partitioning. 8 As a result, multiple consecutive rounds of partitioning are typically used for in vitro selection of protein binders from oligonucleotide libraries. 9−16 In multiround selection of oligonucleotide aptamers, the number of rounds is theoretically unlimited, but a large number of rounds can lead to selection failure due to sequence biases of polymerases used to amplify oligonucleotides. 17−19 In selection of protein binders from DELs, the number of rounds is limited to three or four due to binder loss in partitioning and the inability to amplify the DNA-encoded small molecules by polymerases. 4,7 As a result of inefficiencies in typical multiround selections, 70 percent of attempts to select oligonucleotide aptamers fail, 3 and nonbinders dominate over binders in the enriched DELs. 20 An ultimate solution for these problems would be having a partitioning method that could enrich protein binders to the level of >90% of binders in the binder-enriched library in a single partitioning step. We have recently introduced Ideal-Filter capillary electro-phoresis (IFCE), in which protein-bound and unbound oligonucleotides move in the opposite directions inside the capillary. 21 The condition of IFCE is achieved when the magnitude of the mobility of electroosmotic flow (EOF) is smaller than that of protein−oligonucleotide complexes and larger than that of free oligonucleotides while the signs of the latter two mobilities are negative. 22 The efficiency of IFCE-based partitioning of binders from nonbinders reaches 10 9 , which is 10 7 times greater than those of solid-phase partitioning methods and 10 4 times greater than that of homogeneous partitioning by capillary electrophoresis (CE) in which protein-bound and unbound oligonucleotides move in the same direction. 9−12,23−27 The classical CE-based partitioning is carried out at higher-than-physiological pH and lower-than-physiological ionic strength of the background electrolyte. Advantageously, the conditions of IFCE are achieved at near-physiological values of pH and ionic strength providing greater biological relevance of selected protein binders and suppressed nonspecific binding of the protein with the oligonucleotides and with the capillary surface. Partitioning by IFCE was shown to facilitate the one-step selection of DNA aptamers, and it

Svetlana M. Krylova - One of the best experts on this subject based on the ideXlab platform.

  • Ideal Filter capillary electrophoresis a highly efficient partitioning method for selection of protein binders from oligonucleotide libraries
    Electrophoresis, 2019
    Co-Authors: An T. H. Le, Svetlana M. Krylova, Sergey N Krylov
    Abstract:

    : Selection of affinity ligands for protein targets from oligonucleotide libraries currently involves multiple rounds of alternating steps of partitioning of protein-bound oligonucleotides (binders) from protein-unbound oligonucleotides (nonbinders). We have recently introduced Ideal-Filter capillary electrophoresis (IFCE) for binder selection in a single step of partitioning. In IFCE, protein-binder complexes and nonbinders move inside the capillary in the opposite directions, and the efficiency of their partitioning reaches 109 , i.e., only one of a billion molecules of nonbinders leaks through IFCE while all binders pass through. The condition of IFCE can be satisfied when the magnitude of the mobility of EOF is smaller than that of the protein-binder complexes and larger than that of nonbinders. The efficiency of partitioning in IFCE is 10 million times higher than those of solid-phase-based methods of partitioning typically used in selection of affinity ligands for protein targets from oligonucleotide libraries. Here, we provide additional details on our justification for IFCE development. We elaborate on electrophoretic aspects of the method and define the theoretical range of EOF mobilities that support IFCE. Based on these theoretical results, we identify an experimental range of background electrolyte's ionic strength that supports IFCE. We also extend our interpretation of the results and discuss in-depth IFCE's prospective in practical applications and fundamental studies.

  • determination of the equilibrium constant and rate constant of protein oligonucleotide complex dissociation under the conditions of Ideal Filter capillary electrophoresis
    Analytical Chemistry, 2019
    Co-Authors: An T. H. Le, Svetlana M. Krylova, Sergey N Krylov
    Abstract:

    Ideal-Filter capillary electrophoresis (IFCE) allows selection of protein binders from oligonucleotide libraries in a single step of partitioning in which protein-bound and unbound oligonucleotides move in the opposite directions. In IFCE, the unbound oligonucleotide does not reach the detector, imposing a problem for finding the equilibrium constant (Kd) and rate constant (koff) of protein–oligonucleotide complex dissociation. We report a double-passage approach that allows finding Kd and koff under the IFCE conditions, i.e. near-physiological pH and ionic strength. First, a plug of the protein–oligonucleotide equilibrium mixture passes to the detector in a pressure-driven flow, allowing for both the complex and free oligonucleotide to be detected as a single first peak. Second, the pressure is turned off and the voltage is applied to reverse the migration of only the complex which is detected as the second peak. The experiment is repeated with a lower voltage consequently resulting in longer travel time...

  • Ideal Filter capillary electrophoresis ifce facilitates the one step selection of aptamers
    Angewandte Chemie, 2019
    Co-Authors: An T. H. Le, Svetlana M. Krylova, Mirzo Kanoatov, Shrey Desai, Sergey N Krylov
    Abstract:

    : Selection of aptamers from oligonucleotide libraries currently requires multiple rounds of alternating steps of partitioning of binders from nonbinders and enzymatic amplification of all collected oligonucleotides. Herein, we report a highly practical solution for reliable one-step selection of aptamers. We introduce partitioning by Ideal-Filter capillary electrophoresis (IFCE) in which binders and nonbinders move in the opposite directions. The efficiency of IFCE-based partitioning reaches 109 , which is ten million times higher than that of typical solid-phase partitioning methods. One step of IFCE-based partitioning is sufficient for the selection of a high-affinity aptamer pool for a protein target. Partitioning by IFCE promises to become an indispensable tool for fast and robust selection of binders from different types of oligonucleotide libraries.

  • Determination of the Equilibrium Constant and Rate Constant of Protein–Oligonucleotide Complex Dissociation under the Conditions of Ideal-Filter Capillary Electrophoresis
    Analytical Chemistry, 2019
    Co-Authors: An T. H. Le, Svetlana M. Krylova, Sergey N. Krylov And
    Abstract:

    Ideal-Filter capillary electrophoresis (IFCE) allows selection of protein binders from oligonucleotide libraries in a single step of partitioning in which protein-bound and unbound oligonucleotides move in the opposite directions. In IFCE, the unbound oligonucleotide does not reach the detector, imposing a problem for finding the equilibrium constant (K d) and rate constant (k off) of protein−oligonucleotide complex dissociation. We report a double-passage approach that allows finding K d and k off under the IFCE conditions, i.e. near-physiological pH and ionic strength. First, a plug of the protein−oligonucleotide equilibrium mixture passes to the detector in a pressure-driven flow, allowing for both the complex and free oligonucleotide to be detected as a single first peak. Second, the pressure is turned off and the voltage is applied to reverse the migration of only the complex which is detected as the second peak. The experiment is repeated with a lower voltage consequently resulting in longer travel time of the complex to the detector, greater extent of complex dissociation, and the decreased area of the second peak. Finally, the peak areas are used to calculate the values of K d and k off. Here we explain theoretical and practical aspects of the double-passage approach, prove its validity quantitatively, and, demonstrate its application to determine K d and k off for an affinity complex between a protein and its DNA aptamer. The double-passage approach for finding K d and k off of protein−oligonucleotide complexes under the IFCE conditions is a perfect complement for IFCE-based selection of protein binders from oligonucleotide libraries. S election of protein binders from oligonucleotide libraries, such as random-sequence oligonucleotide libraries and DNA-encoded libraries (DELs), can provide diverse pools of molecules for development of diagnostic probes and drugs. 1−7 Random-sequence oligonucleotide libraries are used to select aptamers, while DELs are used to select small-molecules, capable of binding target proteins. The low abundance of protein binders in oligonucleotide libraries makes it very hard to separate binders from nonbinders in a single step of partitioning. 8 As a result, multiple consecutive rounds of partitioning are typically used for in vitro selection of protein binders from oligonucleotide libraries. 9−16 In multiround selection of oligonucleotide aptamers, the number of rounds is theoretically unlimited, but a large number of rounds can lead to selection failure due to sequence biases of polymerases used to amplify oligonucleotides. 17−19 In selection of protein binders from DELs, the number of rounds is limited to three or four due to binder loss in partitioning and the inability to amplify the DNA-encoded small molecules by polymerases. 4,7 As a result of inefficiencies in typical multiround selections, 70 percent of attempts to select oligonucleotide aptamers fail, 3 and nonbinders dominate over binders in the enriched DELs. 20 An ultimate solution for these problems would be having a partitioning method that could enrich protein binders to the level of >90% of binders in the binder-enriched library in a single partitioning step. We have recently introduced Ideal-Filter capillary electro-phoresis (IFCE), in which protein-bound and unbound oligonucleotides move in the opposite directions inside the capillary. 21 The condition of IFCE is achieved when the magnitude of the mobility of electroosmotic flow (EOF) is smaller than that of protein−oligonucleotide complexes and larger than that of free oligonucleotides while the signs of the latter two mobilities are negative. 22 The efficiency of IFCE-based partitioning of binders from nonbinders reaches 10 9 , which is 10 7 times greater than those of solid-phase partitioning methods and 10 4 times greater than that of homogeneous partitioning by capillary electrophoresis (CE) in which protein-bound and unbound oligonucleotides move in the same direction. 9−12,23−27 The classical CE-based partitioning is carried out at higher-than-physiological pH and lower-than-physiological ionic strength of the background electrolyte. Advantageously, the conditions of IFCE are achieved at near-physiological values of pH and ionic strength providing greater biological relevance of selected protein binders and suppressed nonspecific binding of the protein with the oligonucleotides and with the capillary surface. Partitioning by IFCE was shown to facilitate the one-step selection of DNA aptamers, and it

Sergey N. Krylov And - One of the best experts on this subject based on the ideXlab platform.

  • Determination of the Equilibrium Constant and Rate Constant of Protein–Oligonucleotide Complex Dissociation under the Conditions of Ideal-Filter Capillary Electrophoresis
    Analytical Chemistry, 2019
    Co-Authors: An T. H. Le, Svetlana M. Krylova, Sergey N. Krylov And
    Abstract:

    Ideal-Filter capillary electrophoresis (IFCE) allows selection of protein binders from oligonucleotide libraries in a single step of partitioning in which protein-bound and unbound oligonucleotides move in the opposite directions. In IFCE, the unbound oligonucleotide does not reach the detector, imposing a problem for finding the equilibrium constant (K d) and rate constant (k off) of protein−oligonucleotide complex dissociation. We report a double-passage approach that allows finding K d and k off under the IFCE conditions, i.e. near-physiological pH and ionic strength. First, a plug of the protein−oligonucleotide equilibrium mixture passes to the detector in a pressure-driven flow, allowing for both the complex and free oligonucleotide to be detected as a single first peak. Second, the pressure is turned off and the voltage is applied to reverse the migration of only the complex which is detected as the second peak. The experiment is repeated with a lower voltage consequently resulting in longer travel time of the complex to the detector, greater extent of complex dissociation, and the decreased area of the second peak. Finally, the peak areas are used to calculate the values of K d and k off. Here we explain theoretical and practical aspects of the double-passage approach, prove its validity quantitatively, and, demonstrate its application to determine K d and k off for an affinity complex between a protein and its DNA aptamer. The double-passage approach for finding K d and k off of protein−oligonucleotide complexes under the IFCE conditions is a perfect complement for IFCE-based selection of protein binders from oligonucleotide libraries. S election of protein binders from oligonucleotide libraries, such as random-sequence oligonucleotide libraries and DNA-encoded libraries (DELs), can provide diverse pools of molecules for development of diagnostic probes and drugs. 1−7 Random-sequence oligonucleotide libraries are used to select aptamers, while DELs are used to select small-molecules, capable of binding target proteins. The low abundance of protein binders in oligonucleotide libraries makes it very hard to separate binders from nonbinders in a single step of partitioning. 8 As a result, multiple consecutive rounds of partitioning are typically used for in vitro selection of protein binders from oligonucleotide libraries. 9−16 In multiround selection of oligonucleotide aptamers, the number of rounds is theoretically unlimited, but a large number of rounds can lead to selection failure due to sequence biases of polymerases used to amplify oligonucleotides. 17−19 In selection of protein binders from DELs, the number of rounds is limited to three or four due to binder loss in partitioning and the inability to amplify the DNA-encoded small molecules by polymerases. 4,7 As a result of inefficiencies in typical multiround selections, 70 percent of attempts to select oligonucleotide aptamers fail, 3 and nonbinders dominate over binders in the enriched DELs. 20 An ultimate solution for these problems would be having a partitioning method that could enrich protein binders to the level of >90% of binders in the binder-enriched library in a single partitioning step. We have recently introduced Ideal-Filter capillary electro-phoresis (IFCE), in which protein-bound and unbound oligonucleotides move in the opposite directions inside the capillary. 21 The condition of IFCE is achieved when the magnitude of the mobility of electroosmotic flow (EOF) is smaller than that of protein−oligonucleotide complexes and larger than that of free oligonucleotides while the signs of the latter two mobilities are negative. 22 The efficiency of IFCE-based partitioning of binders from nonbinders reaches 10 9 , which is 10 7 times greater than those of solid-phase partitioning methods and 10 4 times greater than that of homogeneous partitioning by capillary electrophoresis (CE) in which protein-bound and unbound oligonucleotides move in the same direction. 9−12,23−27 The classical CE-based partitioning is carried out at higher-than-physiological pH and lower-than-physiological ionic strength of the background electrolyte. Advantageously, the conditions of IFCE are achieved at near-physiological values of pH and ionic strength providing greater biological relevance of selected protein binders and suppressed nonspecific binding of the protein with the oligonucleotides and with the capillary surface. Partitioning by IFCE was shown to facilitate the one-step selection of DNA aptamers, and it

Tong Ye Wang - One of the best experts on this subject based on the ideXlab platform.

  • empirical predictor of conditions that support Ideal Filter capillary electrophoresis
    Electrophoresis, 2020
    Co-Authors: Tong Ye Wang, Liang Hu, Sergey N Krylov
    Abstract:

    : 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.