22
SHIGEYOSHI OBA AND TAKEHIKO KOJI
- Terminal deoxynucleotidyl transferase (TdT), TdT reaction
buffer and CoCL2 (Boehringer Mannheim Biochemicals, Germany)
- Biotin-16-dUTP (Boehringer Mannheim Biochemicals, Germany)
- digoxigenin-ll-dUTP (Boehringer Mannheim Biochemicals,
Germany)
- HRP labeled goat anti-biotin antibody(VectorLab. Inc., U.S.A.)
- HRP labeled sheep anti-digoxigenin antibody (Boehringer
Mannheim Biochemicals, Germany)
- Normal goat IgG (Dako, Copenhagen, Denmark)
- Normal sheep IgG (Dako, Copenhagen, Denmark)
- Bovine serum albumin (BSA)(Sigma chemical Co., U.S.A.)
- Dithiothreitol (DTT) (Sigma chemical Co., U.S.A.)
- deoxyadenosine triphosphate (dATP) (Sigma chemical Co.,
U.S.A.)
- Brij 35 (Sigma chemical Co., U.S.A.)
- 0.075% (v/v) Brij35/PBS; 5ml Brij35/2000ml PBS
Ix TdT buffer (total 1000 /-11)
distilled water
5x reaction buffer (Boehringer)
Terminal transferase kit (Boehringer Mannheim Biochemicals, Germany;
No. 220582) contains 5X reaction buffer, CoCh and TdT
TdT reaction solution (total 1000/-ll)-biotin
distilled water
5X reaction buffer
(Boehringer Industries)
DTT 10 mM
CoC12 25mM (Boehringer Industries)
dATP 1mM
biotin-16-dUTP
(Boehringer Industries, ImM)
TdT(Boehringer Industries, 25U/ml)
10/-11 (O.lmM; final concentration)
60/-11 (1.5mM)
20/-11 (20/-lM)
5/-11 (5/-1M)
Terminal transferase kit (Boehringer Mannheim Biochemicals, Germany;
No. 220582) contains 5Xreaction buffer, CoCh and TdT You may use lower
volume ofbiotin-16-dUTP when there is excessive background staining in
your sample (Fig. 4). You had better stock DTT (lOmM) and dATP(ImM)/
distilled water below -20°C