The purified ADAM17(ECD)-binding mAbs D8P1C1 and D5P2A11, as well as the mAb MED13622, were buffer-exchanged into 25 mM Tris, pH 9.0, containing 2 μM ZnCl2, and 0.005% (w/v) Brij-35. ADAM17-antibody complexes were formed at a 1:1 molar ratio prior to the assay. The assay was carried out by mixing 50 μM of a fluorogenic peptide substrate Mca-PLAQAV-Dpa (R&D Systems Cat# ES003) with ADAM17-antibody complexes at 37 °C and monitoring the progress of the enzymatic reaction by fluorescence emission (excitation 320 nm and emission 405 nm) over a time course of 1 h using a SpectraMax M5. ADAM17(ECD) alone was used as a positive control. The substrate peptide was derived from TNFalpha and contains a highly fluorescent 7-methoxycoumarin group and a quencher group, 2,4-dinitrophenyl. ADAM17 cleaves the amide bond between the fluorescent and the quencher group causing an increase in fluorescence (R&D Systems, Cat Number ES003). [15 (link), 16 ].
Spectramax m5
Manufactured by R&D Systems
The SpectraMax M5 is a multi-mode microplate reader designed for a variety of scientific applications. It is capable of performing absorbance, fluorescence, and luminescence measurements. The device is equipped with a monochromator-based optical system that allows for flexible wavelength selection.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using spectramax m5
1
ADAM17 Enzymatic Activity Assay
2
Phosphate Assay for Glycosyltransferase Activity
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Phosphate
assays were conducted
in a 96-well plate using reagents from a glycosyltransferase activity
kit (R&D Systems).20 (link) Two microliters
of an enzyme reaction mixture was combined with 48 μL of doubly
distilled water (ddH2O), followed by 30 μL of malachite
green reagent A, 100 μL of ddH2O, and 30 μL
of malachite green reagent B. After a 20 min incubation, the absorbance
at 620 nm was measured on a Spectramax M5 microplate reader alongside
five standards of 0.0–1.0 mM KH2PO4 prepared
identically. Mevalonate pyrophosphate decarboxylase from Saccharomyces
cerevisiae, prepared as described previously,15 (link) served as a positive control for the release
of free phosphate through the decarboxylation of (RS)-mevalonate pyrophosphate.
assays were conducted
in a 96-well plate using reagents from a glycosyltransferase activity
kit (R&D Systems).20 (link) Two microliters
of an enzyme reaction mixture was combined with 48 μL of doubly
distilled water (ddH2O), followed by 30 μL of malachite
green reagent A, 100 μL of ddH2O, and 30 μL
of malachite green reagent B. After a 20 min incubation, the absorbance
at 620 nm was measured on a Spectramax M5 microplate reader alongside
five standards of 0.0–1.0 mM KH2PO4 prepared
identically. Mevalonate pyrophosphate decarboxylase from Saccharomyces
cerevisiae, prepared as described previously,15 (link) served as a positive control for the release
of free phosphate through the decarboxylation of (RS)-mevalonate pyrophosphate.
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