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 6α-hydroxylation
CYP2C9 Diclofenac 4′-hydroxylation
CYP2C19 (S)-Mephenytoin 4′-hydroxylation
CYP2D6 Bufuralol 1′-hydroxylation
CYP2E1 Chlorzoxazone 6-hydroxylation
CYP3A4 Testosterone 6β-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 |
