Optimizing Kinase Assays for Ultrahigh-Throughput Profiling using the Kinase-Glo plus Assay

Worzella, Tracy J; Gallagher, Aoife

doi:10.1016/j.jala.2006.07.001

Cited by 10 publications

(10 citation statements)

References 11 publications

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“…The IC 50 values for all extracts toward GSTs, however, were all greater than the highest concentration used (750 μg/ml). For these extracts, an accurate IC 50 could not be determined because maximum inhibition (>70% inhibition) did not occur at higher doses [ 10 ]. Figure 1 .…”

Section: Resultsmentioning

confidence: 99%

In Vitro and in Vivo Effects of Three Different Mitragyna speciosa Korth Leaf Extracts on Phase II Drug Metabolizing Enzymes—Glutathione Transferases (GSTs)

et al. 2010

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In the present study, we investigate the effects of three different Mitragyna speciosa extracts, namely methanolic, aqueous and total alkaloid extracts, on glutathione transferase-specific activity in male Sprague Dawley rat liver cytosol in vitro and in vivo. In the in vitro study, the effect of Mitragyna speciosa extracts (0.01 to 750 µg/mL) against the specific activity of glutathione transferases was examined in rat liver cytosolic fraction from untreated rats. Our data show concentration dependent inhibition of cytosolic GSTs when Mitragyna speciosa extract was added into the reaction mixture. At the highest concentration used, the methanolic extract showed the highest GSTs specific activity inhibition (61%), followed by aqueous (50%) and total alkaloid extract (43%), respectively. In in vivo study, three different dosages; 50, 100 and 200 mg/kg for methanolic and aqueous extracts and 5, 10 and 20 mg/kg for total alkaloid extract were given orally for 14 days. An increase in GST specific activity was generally observed. However, only Mitragyna speciosa aqueous extract with a dosage of 100 mg/kg showed significant results: 129% compared to control.

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Section: Resultsmentioning

confidence: 99%

In Vitro and in Vivo Effects of Three Different Mitragyna speciosa Korth Leaf Extracts on Phase II Drug Metabolizing Enzymes—Glutathione Transferases (GSTs)

et al. 2010

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“…To measure ATPase activity of ClpC1, there are many ATPase assays available, such as based on the amount of free-phosphate released or ADP generated from the reaction. The previous study indicated that ADP-Glo TM reagent, which measures ADP, was relatively insensitive but has the advantage, allowing us to measure low amounts of ADP production by using the slope of the progress curve and compatible with high ATP concentrations (Worzella, Larson, 2006;Cali et al, 2008). While BIOMOL ® GREEN reagent, which is an endpoint assay to measure free-phosphate, can be sensitive in the single-digit micromolar range.…”

Section: Testing the Atpase Activity Of Fluorescence Free -Phosphate mentioning

confidence: 99%

The high-throughput screening system for inhibitor Mycobacterium tuberculosis compounds based on ATP hydrolysis activity of recombinant protein ClpC1

Duc¹,

Lee²,

Suh³

et al. 2020

Vietnam J. Biotechnol.

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The global Tuberculosis (TB) rate continues to increase by 1% per year with the widespread of drug-resistant TB. Therefore, the development and research to find new anti-TB drugs are becoming an extremely urgent mission. To be able to screen lead anti-tuberculosis drugs, currently, researchers have to carry out directly on the cells of Mycobacterium tuberculosis and to be performed in bio-security facilities level 3 or 4, to prevent infection from pathogens. However, our results demonstrated that the screening of anti-TB drug candidates can be implemented in bio-security facilities level 1 laboratory with the Escherichia coli cell extraction and recombinant ClpC1 protein - an integral part of the Mycobacterium tuberculosis genome. We focused on the ATP hydrolysis activity of ClpC1 to create a specific research direction for the high-throughput anti-tuberculosis drug screening system. ClpC1 protein was overexpressed and purified with functionally characterized (93.5 kDa). The steady-state growth of recombinant ClpC1 protein in Luria-Bertani (LB) broth High Salt medium was maintained and stabilized after extraction. The determined ATPase activity of ClpC1 was performed by measuring the released phosphate from the reaction. Ecumicin was chosen to be a control compound with expected ATP hydrolysis activities (Hill coefficient = 1,19 ± 0,217; Kd value = 0,52 ± 0,275). We tested this high throughput screening system with ten anti-TB drugs to evaluate the effectiveness of our screening system. Based on these results, we built a complete high-throughput screening system anti-tuberculosis drug safety and quickly.

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“…Auch wenn ein bestimmter Schritt der Signalübertragung in biochemischen Untersuchungsmethoden von einem Wirkstoff blockiert wird, ist es durchaus möglich, dass in der Zelle die Signalübertragung nicht beeinflusst wird, da alternative Signalübertragungswege den Ausfall der blockierten Kinase überbrücken können. Daher ist es wichtig, das Verhalten eines Wirkstoffes auch durch In‐vivo‐Untersuchungen in Zellen oder sogar in Lebewesen zu studieren 11–13. Mit solchen Untersuchungen an ganzen Zellen gelingt es, das Permeationsvermögen und die Verteilung des Wirkstoffs in der Zelle, aber auch seine Toxizität zu beurteilen 11–13.…”

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“…Daher ist es wichtig, das Verhalten eines Wirkstoffes auch durch In‐vivo‐Untersuchungen in Zellen oder sogar in Lebewesen zu studieren 11–13. Mit solchen Untersuchungen an ganzen Zellen gelingt es, das Permeationsvermögen und die Verteilung des Wirkstoffs in der Zelle, aber auch seine Toxizität zu beurteilen 11–13. Daher besteht die Hoffnung, dass auf diese Weise Irrwege in den klinischen Entwicklungsprozessen vermieden werden können.…”

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Rapid on-site evaluation of axillary fine-needle aspiration cytology in breast cancer (Br J Surg 2012; 99: 807–812)

Alex

2012

British Journal of Surgery

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The Editors welcome topical correspondence from readers relating to articles published in the Journal. Responses should be sent electronically via the BJS website (http://www.bjs.co.uk). All letters will be reviewed and, if approved, appear on the website. A selection of these will be edited and published in the Journal. Letters must be no more than 250 words in length. Copyright © 2011 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd.

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Optimizing Kinase Assays for Ultrahigh-Throughput Profiling using the Kinase-Glo plus Assay

Cited by 10 publications

References 11 publications

In Vitro and in Vivo Effects of Three Different Mitragyna speciosa Korth Leaf Extracts on Phase II Drug Metabolizing Enzymes—Glutathione Transferases (GSTs)

In Vitro and in Vivo Effects of Three Different Mitragyna speciosa Korth Leaf Extracts on Phase II Drug Metabolizing Enzymes—Glutathione Transferases (GSTs)

The high-throughput screening system for inhibitor Mycobacterium tuberculosis compounds based on ATP hydrolysis activity of recombinant protein ClpC1

Rapid on-site evaluation of axillary fine-needle aspiration cytology in breast cancer (Br J Surg 2012; 99: 807–812)

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