An Automated High Throughput Filtration Assay: Application to Polymerase Inhibitor Identification

An assay for the Maillard reaction has been developed to screen efficiently inhibitors from natural resources such as extracts of plants. The fluorometric analysis of fluorescent material based on advanced glycation endproducts (AGEs) was applied to measurement of an inhibitory index of the Maillard reaction to detect all of the inhibitors at each step of the complicated reaction. To solve the two major problems, slowness of the reaction rate and the existence of interfering substances such as quencher and fluorescent material in the screening sources, we devised the following procedures. Slowness of the reaction rate was solved by raising the reaction temperature to 60°C at which the conformation of bovine serum albumin (BSA) did not change greatly. To remove the interfering substances, AGEs-BSA was precipitated from the reaction mixture. Thus, an efficient assay system by measuring the fluorescent intensity based on AGEs was established to isolate the glycation inhibitors from natural product extracts.

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“…This method is applicable to high throughput screening using a 96/384 well fluorescence plate reader. [32][33][34] …”

Section: Discussionmentioning

confidence: 99%

Screening System for the Maillard Reaction Inhibitor from Natural Product Extracts.

Matsuura

Aradate

Sasaki

et al. 2002

JOURNAL OF HEALTH SCIENCE

155

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“…It is essential that these problems are understood and resolved. An automated high-throughput filtration assay for the identification of RNA polymerase inhibitors has recently been described (136) and might be applied to inhibitors in RT-PCR. Other drug-discovery assays might be employed for rapid and quantitative screening of many classes of inhibitor.…”

Section: Mechanisms Of Inhibitionmentioning

confidence: 99%

Inhibition and facilitation of nucleic acid amplification

Wilson

1997

Appl Environ Microbiol

1,897

827

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“…Such an assay provides a direct measure of the rate of nucleotide addition, and the results correlate proportionally with results obtained from other promoter-dependent, abortive, and elongation assays. 7,13,14,74,141 The high-throughput assay measures the incorporation of α-32 P-rUTP into trichloroacetic acid (TCA)insoluble transcripts by incubating reaction mixtures containing a cocktail of reagents with aliquots of robotically assembled RNAPs in a 96-well thin wall plate using reaction conditions previously described. 7 Because of the hyperthermophilic origin of the recombinant mjRNAP, 70 these reactions are incubated at 70 °C using a thermocycler integrated on the robotic platform.…”

Section: High-throughput Transcription Assaysmentioning

confidence: 99%

The RNA Polymerase Factory and Archaeal Transcription

Weinzierl

2013

Chem. Rev.

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An Automated High Throughput Filtration Assay: Application to Polymerase Inhibitor Identification

Cited by 15 publications

References 8 publications

Screening System for the Maillard Reaction Inhibitor from Natural Product Extracts.

Screening System for the Maillard Reaction Inhibitor from Natural Product Extracts.

Inhibition and facilitation of nucleic acid amplification

The RNA Polymerase Factory and Archaeal Transcription

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