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rev:September 14, 1996
CYTOCHROME P450 RESEARCH PRODUCTS
available through Research Diagnostics Inc
-Update September 14, 1996: please note that we are reformulating our recombinant enzymes to make them easier to use-please ask to be on our mailing list and we will send updated details-expected availability fall/winter 1996. We are working on supplying natural isolates of the following-please inquire for more details: CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2E1, CYP3A4, CYP4A11, P450 reductase & cytochrome b5.
Anti-Cytochrome P450 Antibodies
(rabbit polyclonal). P450 antibodies give 90% inhibition of prototypic reactions catalyzed by the corresponding Ag in intact liver microsomes. All P450 abs except the CYP2C9 IgGs react specifically with their respective antigens on western blots. CYP2C9 ab cross reacts with other CYP2C proteins in human liver.. Available as purified rabbit IgG fractions (lyophilized). Click on catalog number to view a sample spec sheet .
CATALOG# Price 10% discount for 3 or more any combination
RDI-CYP2A6abr $250.00/1mg
RDI-CYP2C9abr $250.00/mg
RDI-CYP2E1abr $250.00/1mg
RDI-CYP2C19Mabr $650.00/1mg NEW MONOSPECIFIC FORMULATION avail: 8/12/96
RDI-CYP3A4abr (backorder until winter 96)
RDI-NORMCTRL (non immune control serum $100.00/mg)
additional antibodies of interest:
RDI-P450REDabr $325.00/0.1ml anti-cytochrome P450 reductase
RDI-P450SCCabr $300.00/0.1ml anti-cytochrome P450 side chain cleavage enzyme
Suggested ratio for in vitro Characterization of Anti-Human CYP Antibodies (not all antibodies yet available-in production)
Antibody Amt for Immunoblota Amt for Inhibitionb Cross w/other P450's Species cross
CYP3A4 7.5 ug/ml 5 mg/nmole c all CYP3As rat CYP3As
CYP2E1 15 ug/ml 10 mg/nmole none rat, rabbit, hamster
CYP2A6 15 ug/ml 5 mg/nmole none ND
CYP2C9 15 ug/ml 5 mg/nmole 2C8, 2C19 ND
CYP2C19 15 ug/ml 1.5 mg/nmole none ND
CYP4A12 15 ug/ml ND none ND
CYP4F4 15 ug/ml ND none ND
CYP2C8 15 ug/ml ND none ND
a Amount of antibody required for colorimetric visualization of proteins. Titer for each application. Generally less is need for more sensitive detection systems as ECLtm or other chemiluminescence based systems.
b This concentration of antibody produces >90% inhibition when mixed with human liver microsomes and preincubated 3 minutes at 37 DEG C prior to addition of reaction components
c mg/nmole = mg IgG per nmole of human liver microsomal P450. To convert to mg IgG required for >90% inhibition/mg of
microsomal protein, assume 0.3 nmole P450/mg microsomal protein.
ND= not determined
CYTOCHROME P450 Research Products
Suggested Protocol for Standard Reconstitution of our recombinant Cytochrome P450's:
-ref: Dieter, H. and Johnson, E.F. (1982) J. Biol. Chem. 257:9315-9323.
Components: Cytochrome P450
Cytochrome P450 Reductase
Lipid (L-alpha-phosphatidylcholine dilauroyl ( C 12:0) Sigma Chemical cat#P-1263
NADPH
50mM Potassium Phosphate Buffer pH 7.4 (KPi buffer) pH by mixing monobasic and dibasic KPi
Preparation of lipid: A stock solution (3mg/ml) is prepared fresh in KPi buffer. Prior to use, the lipid solution must be dispersed by four 5 second pulses at 20% power using a Branson 200 series sonifier (or similar device) fitted with a microprobe.
Reconstitution: To assess substrate metabolism, combine 10 pmole cytochrome P450 (example: 10ul of a 1 nmole/ml solution) with 0.3 units cytochrome P450 reductase (example 10ul of a 30 unit/ml solution) and 30ug freshly prepared lipid (example 10ul of a 3 mg/ml solution) and incubate on ice for 10 minutes. To this add the substrate and kPi buffer to a final volume of 980ul and mix (in most cases, the final concentration of the substrate solvent should not exceed 1%). Incubate this mixture for 3 minutes at 37 DEG C. The reaction is initiated by the addition of NADPH to a final concentration of 1mM (20ul of a 50 mM stock solution).
Reductase Dilution buffer: 10mM KPi, pH 7.4, 20% glycerol, 0.1Mm EDTA.
Cytochrome P450's and select substrates
Cytochrome P450 2C8: taxol, retinoic acid, benzo{a}pyrene, arachidonic acid, warfarin
Cytochrome P450 2C9: tolbutamide, phenytoin, arachidonic acid, intienilic acid, warfarin
Cytochrome P450 2C18: warfarin
Cytochrome P450 2C19: S-mephenytoin, diazepam, tolbutamide, progesterone, warfarin
Cytochrome P450 2D6: debrisoquine, sparteine, bufuraol, warfarin
CYTOCHROME P450 INHIBITORS
Below are some of the various compounds with known inhibition activity of the cytochrome P450 enzyme. Please contact us for additional compounds. All materials are for in vitro research use only-not for use in diagnostics-not for use in or on humans or animals .
CAT# Description Price
30.152.82 R 41400 A.S. Ketoconazole 99+% 500mg $275.00
(inhibits Cp450c17) Kuhn-Velten et al, Biochem. Pharmacol. 44 (12), 2371 (1992).
30.211.44 R 51211 A.S. Itraconazole 99+% 500mg $275.00
30.191.25 R 18134 A.S. Miconazole 99+% 500mg $275.00
30.182.15 R 14950 A.S. Miconazole Nitrate 99+% 500mg $275.00
RDI-F124 Furafylline 5mg $225.00
(inhibits cp450IA2) Boulenc et al, J. Pharmacol. Exp. Ther. 263 (3), 1471 (1992) & Sesardic et al.,
Br. J. Clin. Pharmacol. 29 (6), 651 (1990)
RDI-S147 Sulfaphenazole, USP grade 5mg $225.00
(inhibits cp4502C enzymes), Leeman et al, Life Sci. 52(1), 29 (1993)
& Yun et al, Cancer Res. 52(7), 1868 (1992).
RDI-EI1105 Proadifen (SKF525A.HCL) 250mg $150.00
(inhibits cytochrome P450 dependent arachidonate metabolism),
T. Galeotti et al. Biochem. Pharmacol. 1983 32, 3285 & K. Kauser et al.
Circulation Res. 1991, 68, 1154 & M.A. Carroll et al. J. Hypertens. 1986, 4, S33.
RDI-EI166 Debrisoquin sulfate (Ro 5-3307) 50mg $150.00
(Useful indicator for cytochrome P450 polymorphism) F.P. Guengerich Trends Pharmacol. Sci.
1989, 10, 107.
RDI-EI169 +_ 4- Hydroxydebrisoquin 10mg $150.00
(Useful indicator for cytochrome P450 polymorphism) D.A.P. Evans et al. J. Med.
Genet. 1980, 17, 102. & L. Daumas et al. J. Chromatography 1991, 570, 89.
CYTOCHROME P450 SUBSTRATES
RDI-T142 Taxol 5mg $150.00
RDI-A203 Arachidonic acid 100mg $200.00
RDI-T136 Tolbutamide, USP grade 5 gram $100.00
RDI-D118 Phenytoin, 10 grams $100.00
RDI-P165 Progesterone 1 gram $100.00
RDI-WARF Warfarintm 25 grams $150.00
RDI-Isoret Isotretinoin 100mg $150.00
RDI-Spart Sparteine salt 10 grams $125.00
Research Diagnostics Inc
Pleasant Hill Road
Flanders NJ 07836
phone (201) 584-7093 or 800-631-9384
EMAIL:ResearchD@aol.com
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