Microsphere‐Based Flow Cytometry Protease Assays for Use in Protease Activity Detection and High‐Throughput Screening

Saunders, Matthew; Edwards, Bruce S.; Zhu, Jingshu; Sklar, Larry A.; Graves, Steven W.

doi:10.1002/0471142956.cy1312s54

Cited by 10 publications

(10 citation statements)

References 25 publications

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“…We have previously described microsphere based HTS assays to detect inhibitors of proteases, anthrax lethal factor (LF) and Botulinum neurotoxin A light chain (BoNT/ALC)(14,25,26). The assays employ recombinant fusion proteins consisting of a protease substrate sequence (peptide with cleavage site) fused at one end with a biotinylated attachment sequence and at the other with green fluorescent protein (GFP).…”

Section: Resultsmentioning

confidence: 99%

“…Protease inhibition assays were performed as previously described(25), but with modifications. Biotinylated GFP protease substrates for LF, BoNTALC and a protease-resistant substrate (pinpointGFP) were prepared and loaded on streptavidin microspheres as previously described (14,25,26).…”

Section: Methodsmentioning

confidence: 99%

“…Biotinylated GFP protease substrates for LF, BoNTALC and a protease-resistant substrate (pinpointGFP) were prepared and loaded on streptavidin microspheres as previously described (14,25,26). Additions to wells were in sequence as follows: 1 st , 4 μL protease buffer; 2 nd , 2 μl of a mixture of 1.5 μM LF and 5 nM BoNTALC in protease buffer; 3 rd , 100 nL of test compounds (1 mM in DMSO); and 4 th , 4 μL containing 2×10 5 /ml of each set of substrate-bearing microspheres.…”

Section: Methodsmentioning

confidence: 99%

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Cluster cytometry for high‐capacity bioanalysis

et al. 2012

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Flow cytometry specializes in high content measurements of cells and particles in suspension. Having long excelled in analytical throughput of single cells and particles, only recently with the advent of HyperCyt sampling technology has flow cytometry’s multi-experiment throughput begun to approach the point of practicality for efficiently analyzing hundreds-of-thousands of samples, the realm of high throughput screening (HTS). To extend performance and automation compatibility we built a HyperCyt-linked Cluster Cytometer platform, a network of flow cytometers for analyzing samples displayed in high-density, 1536-well plate format. To assess performance we used cell and microsphere based HTS assays that had been well characterized in previous studies. Experiments addressed important technical issues: challenges of small wells (assay volumes 10 μL or less, reagent mixing, cell and particle suspension), detecting and correcting for differences in performance of individual flow cytometers, and the ability to reanalyze a plate in the event of problems encountered during the primary analysis. Boosting sample throughput an additional four-fold, this platform is uniquely positioned to synergize with expanding suspension array and cell barcoding technologies in which as many as 100 experiments are performed in a single well or sample. As high-performance flow cytometers shrink in cost and size, cluster cytometry promises to become a practical, productive approach for HTS and other large scale investigations of biological complexity.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Cluster cytometry for high‐capacity bioanalysis

et al. 2012

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“…Whereas previous methods using flow cytometry only allowed the measurement of 10 samples per min, now HyperCyt technology allows analysis of up to 1 sample per second. Methods using flow cytometry as the read-out in screening assays and general principles for applying such systems in multiwell plates have been described previously (15, 16) Recently, assays using beads as a solid support matrix for molecular interactions, have become an industry standard of flow cytometry fluorescence-based measurements (15, 17). This approach has been used to develop a bead-based flow cytometric, fluorescent GTP-binding assay which is highly sensitive and allows real-time measurements (18).…”

Section: Introductionmentioning

confidence: 99%

High-Throughput Flow Cytometry Bead-Based Multiplex Assay for Identification of Rho GTPase Inhibitors

Surviladze

Young

Sklar

2011

Methods in Molecular Biology

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Summary Rho family GTPases and their effector proteins regulate a wide range of cell signaling pathways. In normal physiological conditions their activity is tightly controlled and it is not surprising that their aberrant activation contributes to tumorigenesis or other diseases. For this reason, the identification of small, cell permeable molecules capable of inhibition of Rho GTPases can be extraordinarily useful, particularly if they are specific and act reversibly. Herein we describe a flow cytometric assay, which allows us to measure the activity of six small GTPases simultaneously. GST-tagged small GTPases are bound to six glutathione bead sets each set having a different intensity of red fluorescence at a fixed wavelength. The coated bead sets were washed, combined, and dispensed into 384-well plates with test compounds, and fluorescent-GTP binding was used as the read-out. This multiplex bead-based assay was successfully used for to identify both general and selective inhibitors of Rho family GTPases.

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“…In particular, we varied the target module while keeping the reporter module fixed, enabling detection of multiple targets with a single fluorescent readout. We used this approach for DNA detection by designing five sensors that target corresponding sequences from two plasmids that encode GFP-fusion protein variants, a commercially available Emerald GFP plasmid (referred to as emGFP) and a Pinpoint Xa plasmid containing a SNAP25-GFP fusion protein (referred to as SNAP25) previously developed in our lab [16] . All five sensors used a common reporter module.…”

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A Unified Sensor Architecture for Isothermal Detection of Double‐Stranded DNA, Oligonucleotides, and Small Molecules

et al. 2015

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Pathogen detection is an important problem in many areas of medicine and agriculture, which may involve genomic or transcriptomic signatures, or small molecule metabolites. We report a unified, DNA-based sensor architecture capable of isothermal detection of double-stranded DNA targets, single-stranded oligonucleotides, and small molecules. Each sensor contains independent target detection and reporter modules, enabling rapid design. We detected gene variants on plasmids via a straightforward isothermal denaturation protocol. The sensors were highly specific, even with a randomized DNA background. We achieved a limit of detection of ~15 pM for single-stranded targets and ~5 nM for targets on denatured plasmids. By incorporating a blocked aptamer sequence, we also detected small molecules using the same sensor architecture. This work provides a starting point for multiplexed detection of multi-strain pathogens, and disease states caused by genetic variants (e.g., sickle cell anemia).

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Microsphere‐Based Flow Cytometry Protease Assays for Use in Protease Activity Detection and High‐Throughput Screening

Cited by 10 publications

References 25 publications

Cluster cytometry for high‐capacity bioanalysis

Cluster cytometry for high‐capacity bioanalysis

High-Throughput Flow Cytometry Bead-Based Multiplex Assay for Identification of Rho GTPase Inhibitors

A Unified Sensor Architecture for Isothermal Detection of Double‐Stranded DNA, Oligonucleotides, and Small Molecules

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