By SCHWEITZER, MARY HIGBY
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Generation of Antibody to N ε-(Hexanoyl)lysine We have recently identiﬁed Nε-(hexanoyl)lysine (HEL) as one of the lipid hydroperoxide-derived oxidative modified products (5). We also prepare the polyclonal antibody to the novel lipid-lysine adduct using synthetic hexanoylated KLH as an immunogen. The formation of the antibody recognizing hexanoylated BSA is observed (Fig. 5). The antibody to the hexanoylated KLH recognized the synthetic HEL itself. The antibody to HEL can be directly used for estimation of the oxidative modiﬁcation by the oxidized esteriﬁed fatty acid without an alkaline treatment (ester bond hydrolysis) because HEL is the CH3-terminal derived adduct.
Totowa, NJ 21 22 Borchman and Sinha 4. Lyophylizer. 5. Dry air source for spectrometer chamber (see Note 1). 6. 04, Galactic Industries Corporation, Salem, NH). 7. Vortex Mixer. 8. Paraﬁlm (American National Can, Greenwich CT). 9. Scintilation vial caps (see Note 2). 2. Reagents 1. The following solvents are spectroscopic grade obtained from Fisher Scientiﬁc (Pittsburgh, PA): methanol, n-hexane, isopropanol. 2. Argon (prepuriﬁed). 3. 1. Sample Processing In this study rod outer-segment disk membranes were prepared by differential centrifugation (12) (see Note 3).
The generation of the antibody to 15-HPETE-modiﬁed protein. One month after the immunization of 15-HPETE-KLH into a rabbit, the serum was collected and then used for ELISA. The reactivity of the immunized serum was compared with the serum before immunization (preimmune serum). The immunized serum was reacted with 15-HPETE-BSA in a dose-dependent manner. Fig. 5. The generation of the antibody to hexanoylated protein. One month after the immunization of hexanoylated KLH into a rabbit, the serum was collected and then used for ELISA.