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1. 100 mM ATP stock solution made by adjusting pH with
sodium hydroxide (NaOH) to 7.2, and stored at −80 °C.
2. 20 mM Firefly d-luciferin (LH 2 ) (available from Biosynth,
Staad, Switzerland) stock solution made by adjusting pH by
NaOH to 7.3, and stored at −20 °C.
3. QuikChange Site-Directed Mutagenesis kit and/or QuikChange
Multi Site-Directed Mutagenesis kit (Agilent Technologies,
Santa Clara, CA).
4. In-Fusion HD cloning kit (Takara-Bio, Otsu, Shiga, Japan).
5. E. coli JM109 competent cells.
6. LB agar: 5 g/L yeast extract, 10 g/L tryptone, 10 g/L NaCl
and 15 g/L agar.
7. LB liquid medium: 5 g/L yeast extract, 10 g/L tryptone, and
10 g/L NaCl.
8. LBA agar plate: LB agar supplemented with 100 μg/mL
ampicillin.
9. LBA liquid medium: LB liquid medium supplemented with
100 μg/mL ampicillin.
10. E. coli SHuffle T7 express lysY competent cells (New England
Biolabs Japan, Tokyo, Japan).
11. 10 mM Rapamycin (e.g., LKT Laboratories, St. Paul, MN)
stock solution dissolved in methanol or dimethyl sulfoxide,
stored at −20 °C.
12. 1 M Isopropyl-β-thiogalactopyranoside (IPTG) stock solution
for inducing protein expression.
13. White half area 96-well plate (Costar 3694, Corning-Costar,
Osaka, Japan).
14. A plasmid encoding Photinus pyralis Fluc cDNA such as pGEXPpy or pGEM-luc (Promega, Madison, WI).
15. pET32b (Novagen, Merck, Darmstadt, Germany).
16. TALON metal affinity resin and TALON disposable gravity
column (Clontech, Takara-Bio).
17. Toyopearl HW-40S gel filtration resin (Tosoh, Tokyo, Japan).
18. Synthetic genes coding for binding domains such as E. coli
codon-optimized human FK506-binding protein 12 (FKBP12)
and FKBP-rapamycin-associated protein (FRB), appended with
NcoI/SfiI and NotI sites at the 5′ and 3′ ends, respectively.
19. TALON extraction buffer: 300 mM sodium chloride (NaCl),
50 mM sodium phosphate, adjusted to pH 7.0.
20. TALON elution buffer: 150 mM of imidazole in TALON
extraction buffer.
21. MOPS buffer: 100 mM MOPS, 10 mM MgSO 4 , pH 7.3
(see Notes 1 and 2).
Yuki Ohmuro-Matsuyama and Hiroshi Ueda
1. 100 mM ATP stock solution made by adjusting pH with
sodium hydroxide (NaOH) to 7.2, and stored at −80 °C.
2. 20 mM Firefly d-luciferin (LH 2 ) (available from Biosynth,
Staad, Switzerland) stock solution made by adjusting pH by
NaOH to 7.3, and stored at −20 °C.
3. QuikChange Site-Directed Mutagenesis kit and/or QuikChange
Multi Site-Directed Mutagenesis kit (Agilent Technologies,
Santa Clara, CA).
4. In-Fusion HD cloning kit (Takara-Bio, Otsu, Shiga, Japan).
5. E. coli JM109 competent cells.
6. LB agar: 5 g/L yeast extract, 10 g/L tryptone, 10 g/L NaCl
and 15 g/L agar.
7. LB liquid medium: 5 g/L yeast extract, 10 g/L tryptone, and
10 g/L NaCl.
8. LBA agar plate: LB agar supplemented with 100 μg/mL
ampicillin.
9. LBA liquid medium: LB liquid medium supplemented with
100 μg/mL ampicillin.
10. E. coli SHuffle T7 express lysY competent cells (New England
Biolabs Japan, Tokyo, Japan).
11. 10 mM Rapamycin (e.g., LKT Laboratories, St. Paul, MN)
stock solution dissolved in methanol or dimethyl sulfoxide,
stored at −20 °C.
12. 1 M Isopropyl-β-thiogalactopyranoside (IPTG) stock solution
for inducing protein expression.
13. White half area 96-well plate (Costar 3694, Corning-Costar,
Osaka, Japan).
14. A plasmid encoding Photinus pyralis Fluc cDNA such as pGEXPpy or pGEM-luc (Promega, Madison, WI).
15. pET32b (Novagen, Merck, Darmstadt, Germany).
16. TALON metal affinity resin and TALON disposable gravity
column (Clontech, Takara-Bio).
17. Toyopearl HW-40S gel filtration resin (Tosoh, Tokyo, Japan).
18. Synthetic genes coding for binding domains such as E. coli
codon-optimized human FK506-binding protein 12 (FKBP12)
and FKBP-rapamycin-associated protein (FRB), appended with
NcoI/SfiI and NotI sites at the 5′ and 3′ ends, respectively.
19. TALON extraction buffer: 300 mM sodium chloride (NaCl),
50 mM sodium phosphate, adjusted to pH 7.0.
20. TALON elution buffer: 150 mM of imidazole in TALON
extraction buffer.
21. MOPS buffer: 100 mM MOPS, 10 mM MgSO 4 , pH 7.3
(see Notes 1 and 2).
Yuki Ohmuro-Matsuyama and Hiroshi Ueda