A MicroPLC-Based Approach for Determining Kinase-Substrate Specificity

Wu, Jun; Vajjhala, Surekha; O'Connor, Steve

doi:10.1089/adt.2007.072

Cited by 4 publications

(1 citation statement)

References 35 publications

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“…The crude extract was first separated by CLC. The eluent was mixed on-line with AchE and acetylcholine and products detected by ESI-MS. A special arrangement of CLC, micro parallel liquid chromatography, was used by Wu et al [80,81] for studying protein kinase A. Analyses take place parallel in 24 separation capillaries, which makes the method rapid with high throughput.…”

Section: Clc and Cecmentioning

confidence: 99%

Microscale separation methods for enzyme kinetics assays

Křížek

Kubíčková

2012

Anal Bioanal Chem

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Miniaturization continues to be one of the leading trends in analytical chemistry and one that brings advantages that can be particularly beneficial in biochemical research. Use of a miniaturized scale enables efficient analysis in a short time and requires very small amounts of samples, solvents, and reagents. This can result in a remarkable decrease in costs of enzyme kinetics studies, especially when expensive or rare enzymes and/or substrates are involved. Free zone electrophoresis is without a doubt the most common microscale separation technique for capillary and on-chip enzyme assays. Progress and applications in this field are reviewed frequently whereas other modes of separation, although successfully applied, receive only marginal interest in such publications. This review summarizes applications of less common modes of separation in capillary or chip formats, namely micellar electrokinetic chromatography, liquid chromatography, gel electrophoresis, isoelectric focusing, and isotachophoresis. Because these techniques are based on separation mechanisms different from those of free zone electrophoresis, they can be, and have been, successfully used in cases where zone electrophoresis fails. Advantages and drawbacks of these alternative separation techniques are discussed, as also are the difficulties encountered most often and solutions proposed by different research groups.

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Section: Clc and Cecmentioning

confidence: 99%

Microscale separation methods for enzyme kinetics assays

Křížek

Kubíčková

2012

Anal Bioanal Chem

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Structural insights into regulation of the PEAK3 pseudokinase scaffold by 14-3-3

et al. 2023

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PEAK pseudokinases are molecular scaffolds which dimerize to regulate cell migration, morphology, and proliferation, as well as cancer progression. The mechanistic role dimerization plays in PEAK scaffolding remains unclear, as there are no structures of PEAKs in complex with their interactors. Here, we report the cryo-EM structure of dimeric PEAK3 in complex with an endogenous 14-3-3 heterodimer. Our structure reveals an asymmetric binding mode between PEAK3 and 14-3-3 stabilized by one pseudokinase domain and the SHED domain of the PEAK3 dimer. The binding interface contains a canonical phosphosite-dependent primary interaction and a unique secondary interaction not observed in previous structures of 14-3-3/client complexes. Additionally, we show that PKD regulates PEAK3/14-3-3 binding, which when prevented leads to PEAK3 nuclear enrichment and distinct protein-protein interactions. Altogether, our data demonstrate that PEAK3 dimerization forms an unusual secondary interface for 14-3-3 binding, facilitating 14-3-3 regulation of PEAK3 localization and interactome diversity.

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Evaluation of Micro-Parallel Liquid Chromatography as a Method for HTS-Coupled Actives Verification

Simeonov

Yasgar²,

Klumpp³

et al. 2007

ASSAY and Drug Development Technologies

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The identification of biologically active compounds from high-throughput screening (HTS) can involve considerable postscreening analysis to verify the nature of the sample activity. In this study we evaluated the performance of micro-parallel liquid chromatography (microPLC) as a separation-based enzyme assay platform for follow-up of compound activities found in quantitative HTS of two different targets, a hydrolase and an oxidoreductase. In an effort to couple secondary analysis to primary screening we explored the application of microPLC immediately after a primary screen. In microPLC, up to 24 samples can be loaded and analyzed simultaneously via high-performance liquid chromatography within a specially designed cartridge. In a proof-of-concept experiment for screen-coupled actives verification, we identified, selected, and consolidated the contents of "active" wells from a 1,536-well format HTS experiment into a 384-well plate and subsequently analyzed these samples by a 24-channel microPLC system. The method utilized 0.6% of the original 6-microl 1,536-well assay for the analysis. The analysis revealed several non-biological-based "positive" samples. The main examples included "false" enzyme activators resulting from an increase in well fluorescence due to fluorescent compound or impurity. The microPLC analysis also provided a verification of the activity of two activators of glucocerebrosidase. We discuss the benefits of microPLC and its limitations from the standpoint of ease of use and integration into a seamless postscreen workflow.

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A MicroPLC-Based Approach for Determining Kinase-Substrate Specificity

Cited by 4 publications

References 35 publications

Microscale separation methods for enzyme kinetics assays

Microscale separation methods for enzyme kinetics assays

Structural insights into regulation of the PEAK3 pseudokinase scaffold by 14-3-3

Evaluation of Micro-Parallel Liquid Chromatography as a Method for HTS-Coupled Actives Verification

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