For the screening, 60 nl of compounds from the Tocriscreen Mini library (Tocris Bioscience) were transferred to 1536-well plates. The mixtures of CFP-TNKS2-ARC4 (100 nM) and YFP-TBM (200 nM) or TNKS2 SAM(E897K)-CFP (150 nM) and TNKS2 SAM(Y920A)-YFP (300 nM) were prepared in assay buffer (10 mM Bis–Tris-Propane pH 7.0, 3%(w/v) PEG20,000, 0.01%(v/v) Triton-X100 and 0.5 mM TCEP), and rFRET signal was measured at two excitation wavelengths 410 nm and 430 nm. Protein mixtures were transferred to the plates using Mantis liquid dispenser (Formulatrix).
Mantis liquid dispenser
Manufactured by Formulatrix
Sourced in United Kingdom
The Mantis liquid dispenser is a laboratory instrument designed to accurately and precisely dispense small volumes of liquids. It is capable of handling a wide range of liquid types and volumes, making it a versatile tool for various applications in research and development laboratories.
Automatically generated - may contain errors
Lab products found in correlation
Tocriscreen mini library, by Bio-Techne (1 mentions)
Fda approved drug library, by Enzo Life Sciences (1 mentions)
384 well black low volume polypropylene plates, by Thermo Fisher Scientific (1 mentions)
Spectramax m5, by Molecular Devices (1 mentions)
Thapsigargin tg, by Merck Group (1 mentions)
Multidrop combi liquid dispenser, by Thermo Fisher Scientific (1 mentions)
Cyclopiazonic acid cpa, by Bio-Techne (1 mentions)
Echo acoustic dispenser, by Beckman Coulter (1 mentions)
Infinite m1000 pro, by Tecan (1 mentions)
Marvinsketch, by ChemAxon (1 mentions)
7 protocols using mantis liquid dispenser
1
Screening for TNKS2 Inhibitors using rFRET
2
Screening FDA-Approved Drugs for SARS-CoV-2 Inhibitors
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For the screening, 40 nl of 10 mM compound stocks dissolved in DMSO from the FDA-approved drug library (Enzo Life Sciences) were transferred to 384-well black low-volume polypropylene plates (Fisherbrand). The sample mixture containing 1 μM CFP-SARS-CoV-2 nsp3 macrodomain and 5 μM MARylated YFP-GAP was prepared in assay buffer (10 mM Bis–Tris-Propane pH 7.0, 3%(w/v) PEG20,000, 0.01%(v/v) Triton X-100 and 0.5 mM TCEP) and 20 μl per well were dispensed using Mantis liquid dispenser (Formulatrix). The rFRET signal was determined after a 5-minute incubation time. The sample mixtures in presence or absence of 200 μM ADP-ribose were used as positive and negative controls, respectively.
3
FRET Assay for γTuSC-Spc110 Interaction
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The TB150 buffer (50 mM Tris, pH 7.0, 150 mM KCl) used previously (Lin et al., 2014 (link)) led to high levels of Spc110p-independent FRET, so we used a HEPES buffer (40 mM HEPES, pH 7.5, 1 mM MgCl2, 1 mM EGTA, 5 mM DTT), in which γTuSC was better behaved (Supplemental Figure S3C). Proteins were exchanged into assay buffer (HBn with 10% glycerol, 5 mM DTT, and 0.1% phosphatase inhibitor cocktails 2 and 3) using Zeba desalting spin columns (Pierce, Rockford, IL). For assays with Spc110 dimer and tetramer, γTuSC was in assay buffer with 100 mM KCl, and Spc110 was in assay buffer with 250 mM KCl. Spc110 and γTuSC were combined to give a final KCl concentration of 150 mM. For other assays, proteins were prepared in assay buffer with 150 mM KCl. Reactions were assembled in black, clear-bottom, 384-well plates (3655; Corning, Corning, NY) in assay buffer with 0.1 mg/mL bovine serum albumin using a Mantis liquid dispenser (Formulatrix, Waltham, MA) and mixed by pipetting. Reactions were sealed and incubated for 15 min at 25°C. Fluorescence spectra were recorded with a Spectramax M5 plate reader (Molecular Devices, Sunnyvale, CA) with excitation at 420 nm and emission recorded from 460 to 600 nm in 5-nm steps through a 455-nm long-pass filter. Photomultiplier tube sensitivity was set to automatic, and 100 readings were averaged per well.
4
High-throughput SERCA inhibitor screening
5
High-Throughput FRET Screening Assay
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Experiments were done in 1536-well plates and solutions were transferred using Mantis liquid dispenser (Formulatrix). To evaluate concentrations of the constructs used for screening, the FRET signal of TNKS2 CFP-TNKS2-ARC4 and YFP-TBM as well as SAM(E897K)-CFP and TNKS2 SAM(Y920A)-YFP at increasing concentrations of the fret pairs were tested. The ratio of FRET donor to acceptor was kept constant at 1:2. The FRET donor/acceptor concentrations of 50/100, 100/200, 150/300, 200/400 and 250/500 nM were tested, and for each condition 48 datapoints were collected. Volumes were 5 µl per well.
For the calculation of the Zʹ-factors39 (link), CFP-TNKS2-ARC4 (100 nM) and YFP-TBM (200 nM) or TNKS2 SAM(E897K)-CFP (150 nM) and TNKS2 SAM(Y920A)-YFP (300 nM) were mixed and rFRET signal was measured in the presence and absence of 1 M GdnHCl with 680 replicates for each condition. Volumes were 6 µl per well.
For the calculation of the Zʹ-factors39 (link), CFP-TNKS2-ARC4 (100 nM) and YFP-TBM (200 nM) or TNKS2 SAM(E897K)-CFP (150 nM) and TNKS2 SAM(Y920A)-YFP (300 nM) were mixed and rFRET signal was measured in the presence and absence of 1 M GdnHCl with 680 replicates for each condition. Volumes were 6 µl per well.
6
Fluorescence-Based Ubiquitination Assay
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A 30x ubiquitination mix was prepared and dispensed with Echo acoustic dispenser (Labcyte). Final enzyme concentrations in the reaction were: E1 (Ube1; 12 nM), E2 (Ube2D1; 180 nM), E3 (DTX3L; 500 nM), CFP-Ub (25 nM) and YFP-Ub (350 nM). To initiate the reaction, ubiquitination buffer [5 mM Tris (pH 7.5), 200 µM ATP, 0.5 mM MgCl 2 , 200 µM DTT, (final concentrations)] was added to the plate using Mantis liquid dispenser (Formulatrix). Measurements were performed using the multimode microplate reader TECAN Infinite M1000 PRO or TECAN Spark using a fluorescence top reading modality. Excitation wavelength was set to 410 nm (20 nm bandwidth). The emission reading wavelength was set to 477 nm and 527 nm, both at 10 nm bandwidth. Reading gain was set manually to 80% throughout the study. The number of flashes was set to 50 at 400 Hz frequency. Integration time was 20 µs and 44 ms were set for the settling time. Z-position of the reader was fixed to 24019 µm. For the measurements we used 384-well polypropylene, black, flat-bottom plates (Greiner). Except for the buffer optimisation tests, all measurements were performed in reaction buffer [10 mM HEPES (pH7.5)] in 20 µL. Except for the optimisation tests, reactions were measured right after the addition of ubiquitination buffer and after an incubation of 90 min at room temperature.
7
Ubiquitination Assay for Screening
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As a way to validate the functionality of the assay, we screened 2428 compounds (at 100 µM final concentration) from three libraries of the NCI against the ubiquitination reaction with DTX3L. Compounds were transferred to 384-well plates containing E1 (UBE1; 12 nM), E2 (Ube2D1; 180 nM), E3 (DTX3L, 500 nM), Ub Don (25 nM) and Ub Acc (350 nM). To initiate the reaction, ubiquitination buffer [5 mM Tris (pH 7.5), 200 µM ATP, 0.5 mM MgCl 2 , 200 µM DTT, (final concentrations)] was added to the plate using Mantis liquid dispenser (Formulatrix). Reactions were filled to 20 µL with 10 mM HEPES (pH 7.5) and buffer supplemented with 50 mM EDTA was used as a positive control. During the screening, the rFRET was calculated at two excitation wavelengths (410 nm and 430 nm) and reactions were measured after incubating for 90 min at room temperature. MarvinSketch (Chemaxon) was used for drawing chemical structures.
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