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Antibody Protocols: Inhibition
Assay Protocol: 100
µg human liver microsomal protein (Xenotech LLC product H0610) was preincubated
with antiserum (the volumes were as shown on graphs from individual products)
for 10 minutes at room temperature. The preincubated microsomes were then assayed
using standard conditions and the probe substrates detailed below. Probe
Substrates:
| CYP1A1 | 7-Ethoxyresorufin
O-dealkylation | | CYP1A2 | 7-Ethoxyresorufin
O-dealkylation | | CYP2A6 | Coumarin
7-hydroxylation | | CYP2C8 | Paclitaxel
6a-hydroxylation | | CYP2C9 | Diclofenac
4'-hydroxylation | | CYP2C19 | (S)-Mephenytoin
4'-hydroxylation | | CYP2D6 | Bufuralol
1'-hydroxylation | | CYP2E1 | Chlorzoxazone
6-hydroxylation | | CYP3A4 | Testosterone
6b-hydroxylation | Western
Blotting Protocol: Solutions:
| 2x SDS sample
buffer: | 125
mM Tris-acetate pH 6.8, 4% w/v SDS, 10% v/v 2-mercaptoethanol, 0.002% w/v bromophenol
blue | | PBS: | 137
mM NaCl, 2.68 mM KCl, 8.1 mM Na2HPO4,
1.47 mM KH2PO4 |
| TPBS: | 137
mM NaCl, 2.68 mM KCl, 8.1 mM Na2HPO4,
1.47 mM KH2PO4,
0.02% v/v Tween 20 | | Developing
solution: | 30
mg 4-chloro-1-naphthol dissolved in 10 ml methanol added to 50 ml PBS containing
25 µl hydrogen peroxide | Samples
containing 0.5 pmol recombinant cytochrome P450 or 20 µg human liver microsomal
protein (Xenotech
LLC product H0610) were diluted with an equal volume of 2x SDS sample buffer and
incubated at 100°C for 5 min. Samples were separated by SDS polyacrylamide
gel electrophoresis and electro-blotted onto nitrocellulose membrane. The
membrane was washed for 30 min at room temperature in TPBS and then incubated
for 60 min at room temperature in antiserum diluted in TPBS containing 0.2% w/v
bovine serum albumin. Dilutions for each antibody are detailed in the table below. The
membrane was washed three times for 5 min at room temperature in TPBS and incubated
30 min at room temperature in horseradish peroxidase conjugated goat anti-rabbit
IgG (H+L) (Bio-Rad catalogue no. 170-6515) diluted 1:3000 v/v in TPBS containing
0.2% w/v bovine serum albumin. The
membrane was washed three times for 5 min at room temperature in TPBS and incubated
in developing solution at room temperature until specific staining was visible. Antibody
Dilutions: | Antibody | Dilution
in TPBS containing
0.2% w/v
bovine serum albumin (v/v) | | Anti-Human
CYP1A2 | 1:5,000
- 1:10,000 | | Anti-Human
CYP2A6 | 1:5,000
- 1:10,000 | | Anti-Human
CYP2C8 | 1:10,000
- 1:20,000 | | Anti-Human
CYP2C9 | 1:5,000
- 1:10,000 | | Anti-Human
CYP2D6 | 1:5,000
- 1:10,000 | | Anti-Human
CYP3A4 | 1:5,000
- 1:10,000 | | |
