6′-Amino-D-luciferin

6′-Amino-D-luciferin (0.5 mg/mL; 2 min) causes a red shift in the bioluminescence spectrum of green-yellow light-emitting luciferases and a blue shift in the spectrum of red light-emitting luciferases in recombinant beetle luciferase assays, while the spectrum of the 5,5-dimethyl analog is almost identical to that of aminoluciferin^[1].
6′-Amino-D-luciferin (100 μM; 20 μL; 10-40 min) produces a total luminescence output approximately 74% that of native luciferin^[2].
N-Z-DEVD-aLuc (1-100 μM; 45 min) increases caspase-3 activity; at 10 μM, this effect can be completely inhibited by Z-VAD-fmk (HY-16658B)^[3].
6′-Amino-D-luciferin is suitable for IVIS imaging in cell and animal experiments and can be used to analyze interactions with mutant active sites (λmax=588 nm).

Guidelines (The following is our recommended protocol. This protocol is provided for reference only and should be modified according to your specific needs).
1. Enzyme Activity and Kinetic Assays
In vitro experiments^[1]:
Reaction system: 5-10 μL ATP/MgSO₄ mixture (40 mM/80 mM) + 10 μL enzyme solution (0.5-1 mg/mL) + 80-85 μL 0.5 mM 6′-Amino-D-luciferin (pH 8.0 Tris-HCl); bioluminescence intensity is measured at 22°C.
Cell experiments^[2]:
FLuc+ cells (MDA-MB-468): seed 6×10⁴ cells/well, add 100 μM 6′-Amino-D-luciferin, and monitor luminescence kinetics using an IVIS200 imaging system (10-second integration, no filter).

2. Bioluminescence Spectral Analysis
In vitro spectroscopy^[2]:
40 μL PBS + 20 μL ATP buffer (0.5 mM ATP, 0.5 mM CoA, 10 mM MgSO₄) + 1 μL substrate (1.5 mM) + 5 μL FLuc (130 μg/mL). Spectra are collected using a FluoroMax-3 fluorometer (excitation light path blocked, 1 nm increments, 10 nm emission slit width).
In vivo imaging^[2]:
FLuc+ tumor cells are subcutaneously implanted into nude mice, followed by tail vein injection of 2.5 mM 6′-Amino-D-luciferin (100 μL). Imaging is performed using an IVIS200 system (30-second integration, no filter), and tumor signals are normalized.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.