154
4. Transfer buffer: 25 mM Tris–HCl, 192 mM l-glycine.
5. Tris buffered saline (TBS): 50 mM Tris–HCl, 150 mM NaCl,
pH 7.4.
6. TBS with Tween (TBST): TBS supplemented with 0.05% of
Tween 20.
7. TBST with skim milk (TBST-S): TBS-T supplemented with 5%
skim milk.
8. HRP-conjugated anti-His 6 antibody (e.g., Roche Diagnostics
Japan, Tokyo, Japan).
9. Amersham ECL Prime (GE Healthcare, UK).
10. Chemiluminescence
imager
(e.g.,
LAS-4000
mini
Luminoimager, Fujifilm, Tokyo, Japan).
1. Transparent 96-well microplates (e.g., Greiner 655,061).
2. 10 μg/mL streptavidin (e.g., type II, Wako, Osaka, Japan).
3. Phosphate buffered saline (PBS): 1.47 mM KH 2 PO 4 , 8.1 mM
Na 2 HPO 4 , 2.7 mM KCl, 137 mM NaCl, pH 7.4.
4. PBST: PBS containing 0.1% Tween 20.
5. Blocking solution (e.g., Immunoblock, DS Pharma, Osaka, Japan).
6. 1 μg/mL N-terminally biotinylated BGP-C11 peptide in PBST.
7. TMBZ reaction buffer: 100 μg/mL TMBZ (3,3′,5,5′-tetramethylbenzidine), 0.006% H 2 O 2 , 100 mM NaOAc, pH 6.0.
8. 10% H 2 SO 4 to stop the reaction.
1. Black 96-well microplates (e.g., Corning Costar 3693).
2. Transparent 96-well microplates (e.g., Greiner 655061).
3. Dimethyl sulfoxide (DMSO).
4. Fluorocillin™ Green 495/525 β-Lactamase Substrate, soluble
product (Thermo Fisher Scientific, Waltham, MA). 1 mM
stock solution in DMSO can be stored for several months
at −30 °C.
5. Nitrocefin (e.g., Calbiochem, Darmstadt, Germany). 20 mM
stock solution in DMSO can be stored for several months
at −30 °C.
6. PBS containing 1% Triton X-100.
7. PBS containing 3 M urea.
8. PBS containing 20 mg/mL bovine serum albumin (BSA)
(e.g., Sigma-Aldrich, St. Lois, MO).
9. A fluorescence microplate reader (e.g., GENios Pro Tecan, San
Jose, CA).
10. A microplate reader (e.g., SH-1000, Corona Electric, Hitachi,
Ibaraki, Japan).
2.5 ELISA
for Clampbodies
2.6 Enzyme Assays
for Clampbodies
Hiroto Iwai et al.
4. Transfer buffer: 25 mM Tris–HCl, 192 mM l-glycine.
5. Tris buffered saline (TBS): 50 mM Tris–HCl, 150 mM NaCl,
pH 7.4.
6. TBS with Tween (TBST): TBS supplemented with 0.05% of
Tween 20.
7. TBST with skim milk (TBST-S): TBS-T supplemented with 5%
skim milk.
8. HRP-conjugated anti-His 6 antibody (e.g., Roche Diagnostics
Japan, Tokyo, Japan).
9. Amersham ECL Prime (GE Healthcare, UK).
10. Chemiluminescence
imager
(e.g.,
LAS-4000
mini
Luminoimager, Fujifilm, Tokyo, Japan).
1. Transparent 96-well microplates (e.g., Greiner 655,061).
2. 10 μg/mL streptavidin (e.g., type II, Wako, Osaka, Japan).
3. Phosphate buffered saline (PBS): 1.47 mM KH 2 PO 4 , 8.1 mM
Na 2 HPO 4 , 2.7 mM KCl, 137 mM NaCl, pH 7.4.
4. PBST: PBS containing 0.1% Tween 20.
5. Blocking solution (e.g., Immunoblock, DS Pharma, Osaka, Japan).
6. 1 μg/mL N-terminally biotinylated BGP-C11 peptide in PBST.
7. TMBZ reaction buffer: 100 μg/mL TMBZ (3,3′,5,5′-tetramethylbenzidine), 0.006% H 2 O 2 , 100 mM NaOAc, pH 6.0.
8. 10% H 2 SO 4 to stop the reaction.
1. Black 96-well microplates (e.g., Corning Costar 3693).
2. Transparent 96-well microplates (e.g., Greiner 655061).
3. Dimethyl sulfoxide (DMSO).
4. Fluorocillin™ Green 495/525 β-Lactamase Substrate, soluble
product (Thermo Fisher Scientific, Waltham, MA). 1 mM
stock solution in DMSO can be stored for several months
at −30 °C.
5. Nitrocefin (e.g., Calbiochem, Darmstadt, Germany). 20 mM
stock solution in DMSO can be stored for several months
at −30 °C.
6. PBS containing 1% Triton X-100.
7. PBS containing 3 M urea.
8. PBS containing 20 mg/mL bovine serum albumin (BSA)
(e.g., Sigma-Aldrich, St. Lois, MO).
9. A fluorescence microplate reader (e.g., GENios Pro Tecan, San
Jose, CA).
10. A microplate reader (e.g., SH-1000, Corona Electric, Hitachi,
Ibaraki, Japan).
2.5 ELISA
for Clampbodies
2.6 Enzyme Assays
for Clampbodies
Hiroto Iwai et al.