High-Throughput Assay of Oxygen Radical Absorbance Capacity (ORAC) Using a Multichannel Liquid Handling System Coupled with a Microplate Fluorescence Reader in 96-Well Format

2002 ◽  
Vol 50 (16) ◽  
pp. 4437-4444 ◽  
Author(s):  
Dejian Huang ◽  
Boxin Ou ◽  
Maureen Hampsch-Woodill ◽  
Judith A. Flanagan ◽  
Ronald L. Prior
Keyword(s):  
High Throughput ◽  
Oxygen Radical ◽  
Oxygen Radical Absorbance Capacity ◽  
Handling System ◽  
Liquid Handling ◽  
High Throughput Assay

scholarly journals Semi-automated high-throughput substrate screening assay for nucleoside kinases

2021 ◽  
Author(s):  
Katja Hellendahl ◽  
Maryke Fehlau ◽  
Sebastian Hans ◽  
Peter Neubauer ◽  
Anke Kurreck
Keyword(s):  
High Throughput ◽  
Viral Infections ◽  
Screening Assay ◽  
Liquid Handling ◽  
Wide Range ◽  
High Throughput Assay ◽  
Liquid Handling Robot

Nucleoside kinases (NKs) are key enzymes involved in the in vivo phosphorylation of nucleoside analogues used as drugs to treat cancer or viral infections. Having different specificities, the characterization of NKs is essential for drug design and the production of nucleotide analogues in an in vitro enzymatic process. Therefore, a fast and reliable substrate screening assay for NKs is of great importance. Here, we report the validation of a well-known luciferase-based assay for the detection of NK activity in 96-well plate format. The assay was semi-automated using a liquid handling robot. A good linearity was demonstrated (r² >0.98) in the range of 0 to 500 µM ATP, and it was shown that also alternative phosphate donors like dATP or CTP were accepted by the luciferase. The developed high-throughput assay revealed comparable results to HPLC analysis. The assay was exemplary used for the comparison of the substrate spectra of four nucleoside kinases using 20 (8 natural and 12 modified) substrates. The screening results correlated well with literature data and, additionally, previously unknown substrates were identified for three of the NKs studied. Our results demonstrate that the developed semi-automated high-throughput assay is suitable to identify best performing NKs for a wide range of substrates.

scholarly journals High-throughput Quant-iT PicoGreen assay using an automated liquid handling system

BioTechniques ◽  
2019 ◽  
Vol 66 (6) ◽  
pp. 290-294 ◽  
Author(s):  
Kay Anantanawat ◽  
Nicola Pitsch ◽  
Caroline Fromont ◽  
Caroline Janitz
Keyword(s):  
High Throughput ◽  
Handling System ◽  
Liquid Handling ◽  
Picogreen Assay

scholarly journals A High-Throughput Screen for Directed Evolution of the Natural Product Sulfotransferase LipB

2011 ◽  
Vol 16 (8) ◽  
pp. 845-851 ◽  
Author(s):  
Irina Koryakina ◽  
Jessica Neville ◽  
Koichi Nonaka ◽  
Steven G. Van Lanen ◽  
Gavin J. Williams
Keyword(s):  
Directed Evolution ◽  
High Throughput ◽  
Coli Strain ◽  
Liquid Handling ◽  
Cell Extracts ◽  
Acceptor Specificity ◽  
Handling Device ◽  
Suitable Standard ◽  
Small Molecule Therapeutics ◽  
High Throughput Assay

In this article, the authors describe a colorimetric, high-throughput assay suitable for optimizing the activity of the recently discovered sulfotransferase LipB, by directed evolution. Crucially, LipB uses para-nitrophenol sulfate as donor in the sulfation of the nucleoside antibiotic liposidomycin B-I and other acceptor surrogates. Thus, using a robotic liquid-handling device, crude cell extracts were prepared from an Escherichia coli strain that overproduced LipB in wells of a microplate, and production of para-nitrophenol at 405 nm was monitored spectrophotometrically. Enzyme activity could be detected only in the presence of both LipB substrates and overexpressed LipB. The screen displays a suitable standard deviation for directed evolution and importantly is not limited to the natural desulfo-liposidomycin acceptor. The authors plan to use the screen to identify LipB variants with altered acceptor specificity and promiscuity for use in sulfation of natural products and other small-molecule therapeutics.

scholarly journals Fully Automated Peptide Mapping Protocol for Multi-Attribute Method by Liquid Chromatography–Tandem Mass Spectroscopy with a High-Throughput Robotic Liquid Handling System

2020 ◽  
Author(s):  
Chen Qian ◽  
Ben Niu ◽  
Rod Brian Jimenez ◽  
Jihong Wang ◽  
Methal Albarghouthi ◽  
...  
Keyword(s):  
Liquid Chromatography ◽  
Mass Spectroscopy ◽  
High Throughput ◽  
Peptide Mapping ◽  
Tandem Mass ◽  
Handling System ◽  
Liquid Handling ◽  
Hands On ◽  
Liquid Chromatography Tandem Mass ◽  
Tandem Mass Spectroscopy

ABSTRACTThe multi-attribute method (MAM) based on liquid chromatography–tandem mass spectroscopy is emerging as a powerful tool to directly monitor multiple product quality attributes simultaneously. Preparation of samples for MAM, however, is labor intensive, involving protein denaturation, disulfide bond reduction, free cysteine alkylation, and enzymatic digestion steps, which require significant analyst hands-on time while limiting result turnaround. Such complexity can also render nontrivial variations across analysts and laboratories. We describe the development of a fully automated peptide mapping procedure with a high-throughput robotic liquid handling system to improve sample handling capability and outcome reproducibility while saving analyst hands-on time. The automated procedure is completely hands-free, and setup requires the analyst only to prenormalize the sample concentrations and load buffers and reagents at their designated positions on the robotic deck. The robotic liquid handler performs all the subsequent preparation steps and stores the digested samples on a chiller unit to await retrieval. The convenience and flexibility provided by this automated peptide mapping method provides substantial benefits over manual sample preparation protocols. The optimized, automated procedure showed good reproducibility and results that were comparable to those of the manual procedure with respect to sequence coverage, digestion completeness, and quantification of posttranslational modifications. With this increased throughput, coupled with fast MAM analysis, more comprehensive characterization can be achieved.

scholarly journals Assay Miniaturization for Ultra-High Throughput Screening of Combinatorial and Discrete Compound Libraries: A 9600-Well (0.2 Microliter) Assay System

1998 ◽  
Vol 3 (1) ◽  
pp. 55-62 ◽  
Author(s):  
Kevin R. Oldenburg ◽  
Ji-Hu Zhang ◽  
Tongming Chen ◽  
Anthony Maffia ◽  
Karl F. Blom ◽  
...  
Keyword(s):  
High Throughput ◽  
Combinatorial Chemistry ◽  
Therapeutic Target ◽  
High Throughput Screening ◽  
Detection System ◽  
Screening System ◽  
Handling System ◽  
Liquid Handling ◽  
Compound Libraries ◽  
Plate Design

Combinatorial chemistry has opened a new realm of chemical entities in the search for novel therapeutics. Combinatorial chemistry is currently adding hundreds of thousands of compounds to similar numbers available from years of synthesis by medicinal chemistry. It is not unreasonable to expect that over the next several years, nearly a million compounds will be available for screening against each therapeutic target. The number of potential targets will also be increasing with the advances in genomics. With the increasing number of compounds to be screened against an increasing number of targets, it is becoming increasingly difficult and costly to obtain the required amounts of key biological material needed to screen these compounds. One obvious solution is to miniaturize the assays so that the biological reagent supply doesn't need to increase. To this end, we have developed an ultra-high throughput screening system comprised of a new plate design (9600-well), detection system, and liquid handling system. This new format is capable of performing assays in as little as 0.2 Al. The results obtained from this system compare favorably to those obtained in the standard 96-well format.

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