Table 1. Troubleshooting table for cell-free experiments.

Problem	Cause	Solution
Very low or non-existent signal	Inactive Luciferase enzyme	Run a control experiment by incubating D-luciferin with luciferase enzyme. If no light emission is observed, luciferase might be degraded. Use another batch of luciferase enzyme
	Compromised luciferase buffer	Prepare a fresh luciferase buffer (with freshly added ATP) and control activity by incubating the luciferase enzyme with D-luciferin
	Exposure (integration) time is too short	Increase the exposure time on the bioluminescence reader/imager and repeat experiment again
	Too low concentration of probes	Increase probes concentrations
	Delay between luciferase addition and the beginning of acquisition is too long	Decrease the delay between luciferase addition and acquisition
Low signal to background ratio (no increase in light emission when the probe is incubated with protease enzyme).	Inactive protease enzyme.	Verify enzymatic activity of the protease using commercial colorimetric or fluorescent kit (e.g. for caspase-3 : Sigma-Aldrich : NAc-Asp-Glu-Val-Asp-pNA, ref A2559)
	Quality of protease-specific peptide is compromised	Verify that the peptide probe is pure and not oxidized or degraded. HPLC/MS can be used for analysis
	Low purity of peptide	Very high degree of purity is necessary since traces of Free D- Cysteine can result in unspecific formation of D-luciferin and significantly increase background signal. Verify peptide purity using HPLC and MS. If stored solution was used, prepare a fresh one.