50 μL of 7.5 μM DPP1-3 in HEPES (20 mM, pH = 7.4, 150 mM NaCl, 0.1% Triton X-100) were added to a 384-well black flat bottom plate (Corning) and placed in an Infinite M200 Pro (Tecan) plate reader at 37 °C for 5 min to warm. 25 μL of either HEPES buffer alone or HEPES buffer containing 150 nM APT1 or APT2, was added using a multi-channel pipette, resulting in a final concentration of 5 μM DPP1-3 and 50 nM APT1 or APT2. Fluorescence intensities (λex 490/9 nm, λem 545/20 nm, Gain 70, No. of flashes 25, Integration time 100 μs and Z-position 20000 μm) were measured at 30 s time interval for 20 min. At the end of 20 min kinetic run, emission spectra were obtained (λex 485 nm, λem 510-700 nm, Gain 100, No. of flashes 25, Integration time 100 μs and Z-position 20,000 μm). For the catalytically inactive enzyme experiments, 10-times more APT1(S119A) and APT2(S122A) enzyme were used.
384 well black flat bottom plate
Manufactured by Corning
The 384-well black flat bottom plate is a laboratory equipment used for various applications in life science research. This product provides a standardized multi-well format with a flat bottom design to accommodate different sample types and volumes. The black color of the plate helps to minimize light interference and optimize results in certain assays. The 384-well format allows for high-throughput screening and efficient use of limited sample quantities.
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Lab products found in correlation
3 protocols using 384 well black flat bottom plate
1
Fluorometric Assay for Acyl Protein Thioesterase
2
Fluorometric Assay for Acyl Protein Thioesterase
3
TDP-43 Protein-RNA Binding Assay
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Purified TDP-43 WT and mutants previously purified were serially diluted in a 1:2 ratio in a final range of 0–2 μM protein into a 300 mM NaCl, 10 mM Tris (pH 8.0), 5% glycerol, 5% sucrose, 0.5 mM TCEP buffer solution. The protein dilution was mixed with CLIP34 (IDT) 3′ labeled with FITC (fluorescein isothiocyanate) in a final concentration of 100 nM and added in triplicate in a 384-well black flat bottom plate (Corning) in a total reaction volume of 30 μL protected from light. The anisotropy measurements were performed in a Spectra Max i3 plate reader (Molecular Devices) with excitation and emission wavelengths of 480 and 520 nm, respectively. To assess the binding, the anisotropy data was fitted in a nonlinear fit in 4-parameter logistic model to calculate the apparent IC50,app. The apparent IC50 (IC50,app) is the protein concentration in which 50% of the maximal anisotropy change is observed. Data analysis were performed using GraphPad Prism 9.
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