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. Author manuscript; available in PMC: 2015 Sep 9.
Published in final edited form as: Curr Protoc Chem Biol. 2014 Sep 9;6(3):169–189. doi: 10.1002/9780470559277.ch140047

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.