rev: January 23, 2002
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& Related Antibodies
(anti-Human and others as indicated)
RDI Divison of Fitzgerald Industries Intl offers a wide line of antibodies. Since no one antibody works best for all applications (neutralization, blotting, histochemistry, ELISA, etc), we offer many different types of antibodies to help solve this problem. Please inquire for other applications or types of antibodies not listed below.
Rabbit anti-human Cytochrome P450 CYP2C9
Price: $440.00/1mg vial
Presentation: 1 milligram lyophilized from 100mM potassium phosphate buffer pH 76.4, 250mM KCL and 2.5 micromole thimerosal.
species: rabbit IgG (purified from serum using caprylic acid/ammonium sulfate fractionation.
Specificity has been determining by Western blotting. Anti-human CYP2C9 IgG reacts primarily with CYP2C9 but also recognizes CYP2C19 in human liver microsomes; cross-reaction with CYP2C8 is minimal. The antibody also cross-reacts with the homologous CYP2C proteins in rat, rabbit, and hamster liver microsomes. Specificity with whole liver homogenates or S-9 fractions has not been determined.
Reconstitution: Immunoblots: Reconstitute by adding 0.5ml of Storage: distilled
water to one vial of lyophilized IgG (1mg). Rotate vial gently until powder
dissolves. Add 0.5ml of neat glycerol to obtain a 50% glycerol solution
containing 1mg IgG/ml. Store -20 DEG C.
Immunoinhibition: Reconstitute the antibodies in 100 microliters of the assay buffer being used so that the final concentration of antibody is 10mg/ml. Store at -20 DEG C. Avoid frequent freeze thaw cycles. use approx 5 mg antibody IgG /nmol for 90% inhibition with antibody preincubated for 3 minutes at 37 DEG C prior to reaction with enzyme. Assume approx 0.3nmol P450/mg microsomal protein.
Note: All uses and titers must be optimized for each application.
Sample Protocol: western blot:for full size (15 X 15cm) blots, add 100ug rabbit anti-human CYP2C9 IgG to 15- 20ml of blocking solution (eg., Blotto). This corresponds to 100ul of the 1.0mg IgG/ml solution prepared in 50% glycerol as described above. This amount can be scaled down appropriately with smaller size blots. Incubate overnight at room temperature and then discard antibody solution. Wash blot extensively with blocking solution to remove unbound CYP2C9 antibody and then incubate with an anti-rabbit IgG conjugate (eg. anti- rabbit IgG-alkaline phosphatase or anti- rabbit IgG-biotin.)
In the developers lab, they use a goat anti- rabbit IgG biotin conjugate followed by a complex of Streptavidin-horse radish peroxidase and subsequently staining with 4- chloro-1-napththol/hydrogen peroxide. This system allows good visualization of CYP2C9 with 10ug of human liver microsomal protein applied to the original polyacrylamide gel However, an anti-rabbit IgG-alkaline phosphatase conjugate with chemiluminescent staining should be at least as sensitive.
The CYP2C9 antibody (which reacts with CYP2C9, CYP2C8, and CYP2C19) can be used to detect CYP2C19 on Western blots, provided that a full size gel (12 x 14 cm) is first used to resolve these P450 proteins. A Western made from a full-size gel will give three well-separated immunoreactive protein bands. Minigels do not give adequate resolution, cannot separate CYP2C8 from CYP2C19, and give only two immunoreactive bands on the subsequent blot; one of these bands is a composite of CYP2C8 PLUS CYP2C19.
Lane A = UN504 microsomes (10 µg)
Lane B = Purified CYP2C9 (0.1 µg)
Lane C = UC9303 microsomes (10 µg)
Lane D = UC9406 microsomes (10 µg)
Sample Neutralization Graph
RDI Divison of Fitzgerald Industries Intl
34 Junction Square Drive
Concord MA 01742-3049
phone (978) 371-6446 or (800) 370-2222
fax (978) 371-2266
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