The compounds, acetylated peptide [SGRGK(Ac)GGK(Ac)GLGK(Ac)GGAK(Ac)RHRK-biotin] and purified bromodomain protein were added to the OptiPlateTM-384 plate. Then, streptavidin-coated donor beads and anti-GST AlphaScreening acceptor beads were added. The incubation condition was 25 °C for 1 h using a Thermomixer C (Eppendorf, USA). The blocking efficacy (IC50) values were obtained by 8-point titration (from 0.0096 to 32 μM). The IC50 values of inhibitors were evaluated with GraphPad Prism 7 using normalized and inhibition analysis suites.
Optiplate 384
The OptiPlate-384 is a 384-well microplate designed for high-throughput screening applications. It features a black well bottom and white well walls, optimized for luminescence and fluorescence detection. The plate is made of polystyrene and has dimensions of 128 mm x 86 mm.
Lab products found in correlation
31 protocols using optiplate 384
AlphaScreen Assay for Bromodomain Inhibitors
The compounds, acetylated peptide [SGRGK(Ac)GGK(Ac)GLGK(Ac)GGAK(Ac)RHRK-biotin] and purified bromodomain protein were added to the OptiPlateTM-384 plate. Then, streptavidin-coated donor beads and anti-GST AlphaScreening acceptor beads were added. The incubation condition was 25 °C for 1 h using a Thermomixer C (Eppendorf, USA). The blocking efficacy (IC50) values were obtained by 8-point titration (from 0.0096 to 32 μM). The IC50 values of inhibitors were evaluated with GraphPad Prism 7 using normalized and inhibition analysis suites.
cGMP Quantification Assay Protocol
Tau Aggregate Detection Assay
Sandwich Immunoassays for Tau Quantification
Assay reactions (25 μl) were carried out in OptiPlate-384 microplates (PerkinElmer) that contained 5 μL of analyte at the specified protein concentration, 10 μL of biotinylated capture antibody (final concentration 2 nM) and 10 μL of detection antibody-conjugated acceptor beads (final concentration 20 μg/mL). After overnight incubation at 4°C, 25 μL of streptavidin donor beads were added under subdued light conditions (final concentration 40 μg/mL) and the reactions were incubated at room temperature for 60 min with gentle shaking. The fluorescent signal was detected on an Envision Plate Reader at 615 nm (PerkinElmer).
Evaluating Peptide Binding to Influenza Hemagglutinin
Quantitative Protein Analysis via AlphaLISA
Inhibitory Effects of Peptides on Stx2a Binding
CFTR-NHERF1 Binding Assay Protocol
(amplified luminescent proximity homogeneous
assay) GST Detection Kit (PerkinElmer; Waltham, MA) to study interactions
of purified full-length Flag-WT CFTR and Flag-ΔF508 CFTR with
the GST-NHERF1-PDZ2 domain. All proteins used were of high quality
and purified under nondenaturing conditions (we tested protein interactions
with syntaxin 1A at the N-terminus and protein binding to Azido ATP;
data not shown). In brief, starting with a 100 nM concentration, CFTR
protein (WT and ΔF508) was serially diluted (in 1/2 log dilution
series) in the assay buffer (1x HEPES, 0.1% BSA, 0.05% Tween 20 [v/v],
pH 7.2) containing GST-NHERF1-PDZ-2 (100 nM final concentration) and
biotin-CFTR-C-tail peptide (100 nM final concentration). The resulting
solutions were incubated at room temperature for 30 min. In triplicate,
each sample solution (15 μL) was transferred to a white opaque
384-well microplate (OptiPlate-384; PerkinElmer) into which anti-GST
acceptor beads (5 μL; 20 μg/mL final concentration) were
added and incubated at room temperature for 30 min. Streptavidin donor
beads (5 μL; 20 μg/mL final concentration) were then added
and incubated at room temperature for 2 h. The plate was read on a
FLUOstar-Omega plate reader (Ortenberg, Germany).
Quantifying SMOX Levels in A549 Cells
Screening 5-HT6R Inhibitors via cAMP Assay
5-HT6R using their ability
to inhibit cAMP production induced by 1 μM (EC80)
5-carboxamidotryptamine (5-CT). The level of cAMP was measured in
1321N1 cells expressing the h5-HT6R (PerkinElmer,
#ES-316-CF). According to the manufacturer’s instructions,
total cAMP was measured using the LANCE cAMP detection kit (PerkinElmer,
#TRF0263). Cells were incubated with a mixture of compounds for 30
min at room temperature (RT) in a white polystyrene OptiPlate-384
(PerkinElmer, #6007299) microplate. After incubation, the reaction
cells were lysed by the addition of 10 μL of cAMP detection
buffer, including Eu-cAMP tracer and ULight-anti-cAMP working solution.
The plate was incubated at RT for 1 h before measuring the signal
with a Tecan multimode plate reader (Infinite M1000 Pro). Compounds
were tested in triplicate at eight concentrations in the range from
10–11 to 10–4 M. Kb constants were calculated from Cheng–Prusoff
equation63 (link) adapted to functional assays.
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