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for the firefly luciferase substrates, and then for the Renilla
luciferase substrates.
14. If using the self-made substrates for the Mithras device from
Berthold Technologies, a different protocol for measuring the
Renilla luciferase signals will be applied, as this substrate has a
decreased stability. The substrate is injected, followed by an
orbital shake of 2 s, and then immediately measured for 2 s.
This Process is repeated well by well. The detailed protocol for
making both firefly (a) and Renilla (b) substrates is listed
below. We regularly order special reagents from p.j.k. GmbH,
Kleinblittersdorf, Germany; these include coelenterazine,
D-luciferin, co-enzyme A, ATP, DTT (1,4 dithiothreitol).
c(0.001, 0.01, 0.1, 1, 10, 100),
(a) Substrate for firefly luciferase
Chemical
Final concentration
Tricine
20 mM
(MgCO 3 ) 4 *Mg(OH) 2 *5H 2 O
(magnesium
carbonate-hydroxide-pentahydrate)
1.07 mM
MgSO 4 * 7 H 2 O
2.67 mM
EDTA
0.1 mM
DTT
33.3 mM
Coenzym A
270 μM
d-Luciferin, free acid
470 μM
ATP
530 μM
To solve magnesium carbonate, adjust the pH value using
HCl (37%, use approximately 1 ml for 1 l) until solution
becomes clear. Then, adjust the pH value to 7.8 using 5 M
NaOH (use approximately 7.5 ml for 1 l).
(b) Substrate for Renilla luciferase
Chemical
Final concentration
NaCl
1.1 M
Na 2 -EDTA
2.2 mM
K x PO 4 (pH 5,1)
0.22 M
BSA
0.44 mg/ml
NaN 3
1.3 mM
Coelenterazine (solved in EtOH) 1.43 mM
Jan P. Wintgens et al.