Use of protein–protein interactions in affinity chromatography

Muronetz, Vladimir I.; Шолух, М. В.; Korpela, Timo

doi:10.1016/s0165-022x(01)00187-7

Cited by 23 publications

(7 citation statements)

References 62 publications

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“…Multiple methods are utilized to analyze protein-protein interactions, such as pull-down assays, affinity chromatography, NMR spectroscopy, isothermal titration calorimetry, and FRET (Campoy et al, 2004;Gerace & Moazed, 2015;Liu et al, 2016;Muronetz et al, 2001). In this study, we confirmed the interaction between F-actin and gelsolin using fluorescence imaging and FRET analysis.…”

Section: Actin Fiber Changes Induced By Electrical Stimulationsupporting

confidence: 72%

Electric fields regulate cellular elasticity through intracellular Ca²⁺ concentrations

Han

Noh²,

Jang³

et al. 2021

Journal Cellular Physiology

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Cellular elasticity is a key factor related to a broad range of physiological and pathological processes. The elasticity of a single cell has thus emerged as a potential biomarker to characterize the cellular state. Both internal and external stimuli affect cellular elasticity, and changes in elasticity can cause alterations in cellular characteristics or function. The application of electric fields (EFs) is a promising method that can be used to change cellular elasticity; however, the mechanisms underlying its effect remain unknown. Here, we demonstrate EFs-induced elasticity changes in human dermal fibroblasts and discuss the underlying mechanism related to actin polymerization. Cellular elasticity increases after EF (50 mV/mm) stimulation, reaching a maximum at 30 min before decreasing between 30 and 120 min. The cellular elasticity under EF stimulation, regardless of stimulation time, is higher than that of the control. F-actin regulates the elasticity of cells through gelsolin activation. We show changes in intracellular Ca 2+ caused by EFs, which induced gelsolin activation and F-actin content changes. This result demonstrates a series of processes in which external electrical stimulation conditions regulate cellular elasticity.

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Section: Actin Fiber Changes Induced By Electrical Stimulationsupporting

confidence: 72%

Electric fields regulate cellular elasticity through intracellular Ca²⁺ concentrations

Han

Noh²,

Jang³

et al. 2021

Journal Cellular Physiology

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“…Thus, the great interest of detecting protein-protein interactions for proteomic development becomes evident. While the detection of strong protein-protein interactions may be efficiently achieved using a variety of methods (such as affinity chromatography, cross-linking, gel filtration, native and twodimensional electrophoresis) [17][18][19], the detection of very weak interactions remains very difficult. The protein-protein complexes formed by these interactions may be in the minority compared with the isolated components, as these are in Scheme 1.…”

Section: Introductionmentioning

confidence: 99%

Purification, stabilization, and concentration of very weak protein-protein complexes: Shifting the association equilibrium via complex selective adsorption on lowly activated supports

et al. 2005

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Very weak protein-protein interactions are very difficult to detect because these complexes could be under the detection limit or they tend to dissociate. Here, using as a model the antibody-antigen interaction weaken by the presence of dioxane, we have shown a strategy for the protein complexes purification by selective adsorption of the associated proteins. This strategy is based on the use of poorly activated anionic exchanger supports to selectively adsorb large complexes. This selective adsorption of the associated proteins shifted the association equilibrium of the soluble proteins toward the associated form. Thus, in the presence of 15% v/v dioxane, a concentration that is able to almost fully break the immunocomplex (less that 3% of the immunocomplex appeared associated when soluble antigen-antibody mixture was cross-linked with aldehyde-dextran), we can obtain more than 90% of the fully pure immunocomplex from the non-associated protein, adsorbed on anionic exchanger supports having a very low activation. This simple strategy may be a very useful tool to solve one of the most relevant challenges in the modern proteomics, the detection of very weak protein-protein interactions.

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“…The use of solid-supported proteins to evaluate protein-protein interactions and to purify proteins is a well-founded tool (Muronetz et al, 2001; Perret and Boschetti, 2018). Interactions between proteins and small molecules have also been well-explored (de Moraes et al, 2014a; Zheng et al, 2014; Hage, 2017).…”

Section: Introductionmentioning

confidence: 99%

Solid-Supported Proteins in the Liquid Chromatography Domain to Probe Ligand-Target Interactions

2019

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Ligand-target interactions play a central role in drug discovery processes because these interactions are crucial in biological systems. Small molecules-proteins interactions can regulate and modulate protein function and activity through conformational changes. Therefore, bioanalytical tools to screen new ligands have focused mainly on probing ligand-target interactions. These interactions have been evaluated by using solid-supported proteins, which provide advantages like increased protein stability and easier protein extraction from the reaction medium, which enables protein reuse. In some specific approaches, precisely in the ligand fishing assay, the bioanalytical method allows the ligands to be directly isolated from complex mixtures, including combinatorial libraries and natural products extracts without prior purification or fractionation steps. Most of these screening assays are based on liquid chromatography separation, and the binding events can be monitored through on-line or off-line methods. In the on-line approaches, solid supports containing the immobilized biological target are used as chromatographic columns most of the time. Several terms have been used to refer to such approaches, such as weak affinity chromatography, high-performance affinity chromatography, on-flow activity assays, and high-performance liquid affinity chromatography. On the other hand, in the off-line approaches, the binding event occurs outside the liquid chromatography system and may encompass affinity and activity-based assays in which the biological target is immobilized on magnetic particles or monolithic silica, among others. After the incubation step, the supernatant or the eluate from the binding assay is analyzed by liquid chromatography coupled to various detectors. Regardless of the selected bioanalytical approach, the use of solid supported proteins has significantly contributed to the development of automated and reliable screening methods that enable ligands to be isolated and characterized in complex matrixes without purification, thereby reducing costs and avoiding time-laborious steps. This review provides a critical overview of recently developed assays.

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Use of protein–protein interactions in affinity chromatography

Cited by 23 publications

References 62 publications

Electric fields regulate cellular elasticity through intracellular Ca²⁺ concentrations

Electric fields regulate cellular elasticity through intracellular Ca²⁺ concentrations

Purification, stabilization, and concentration of very weak protein-protein complexes: Shifting the association equilibrium via complex selective adsorption on lowly activated supports

Solid-Supported Proteins in the Liquid Chromatography Domain to Probe Ligand-Target Interactions

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Use of protein–protein interactions in affinity chromatography

Cited by 23 publications

References 62 publications

Electric fields regulate cellular elasticity through intracellular Ca2+ concentrations

Electric fields regulate cellular elasticity through intracellular Ca2+ concentrations

Purification, stabilization, and concentration of very weak protein-protein complexes: Shifting the association equilibrium via complex selective adsorption on lowly activated supports

Solid-Supported Proteins in the Liquid Chromatography Domain to Probe Ligand-Target Interactions

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Electric fields regulate cellular elasticity through intracellular Ca²⁺ concentrations

Electric fields regulate cellular elasticity through intracellular Ca²⁺ concentrations