Specificity and cross-reactivity of monoclonal and polyclonal antibodies against cytochrome P-450E of the marine fish scup

Kloepper-Sams, Pamela J.; Park, Sang S.; Gelboin, Harry V.; Stegeman, John J.

doi:10.1016/0003-9861(87)90660-6

Cited by 132 publications

(37 citation statements)

References 25 publications

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“…Equivalent volumes of lysate were subjected to SDS-PAGE and blotted to nitrocellulose. Blots were probed with monoclonal antibody 1-12-3 (3 g/mL) against Stenotomus chrysops (scup) CYP1A (Park et al, 1986;Kloepper-Sams et al, 1987). Blots were subsequently probed with goat anti-mouse IgG horseradish peroxidase (Bio-Rad, Hercules, CA) secondary antibody (1:1000 dilution).…”

Section: Western Blot Analysismentioning

confidence: 99%

Development of the morpholino gene knockdown technique in Fundulus heteroclitus: A tool for studying molecular mechanisms in an established environmental model

et al. 2008

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a b s t r a c t A significant challenge in environmental toxicology is that many genetic and genomic tools available in laboratory models are not developed for commonly used environmental models. The Atlantic killifish (Fundulus heteroclitus) is one of the most studied teleost environmental models, yet few genetic or genomic tools have been developed for use in this species. The advancement of genetic and evolutionary toxicology will require that many of the tools developed in laboratory models be transferred into species more applicable to environmental toxicology. Antisense morpholino oligonucleotide (MO) gene knockdown technology has been widely utilized to study development in zebrafish and has been proven to be a powerful tool in toxicological investigations through direct manipulation of molecular pathways. To expand the utility of killifish as an environmental model, MO gene knockdown technology was adapted for use in Fundulus. Morpholino microinjection methods were altered to overcome the significant differences between these two species. Morpholino efficacy and functional duration were evaluated with molecular and phenotypic methods. A cytochrome P450-1A (CYP1A) MO was used to confirm effectiveness of the methodology. For CYP1A MO-injected embryos, a 70% reduction in CYP1A activity, a 86% reduction in total CYP1A protein, a significant increase in ␤-naphthoflavone-induced teratogenicity, and estimates of functional duration (50% reduction in activity 10 dpf, and 86% reduction in total protein 12 dpf) conclusively demonstrated that MO technologies can be used effectively in killifish and will likely be just as informative as they have been in zebrafish.

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Section: Western Blot Analysismentioning

confidence: 99%

Development of the morpholino gene knockdown technique in Fundulus heteroclitus: A tool for studying molecular mechanisms in an established environmental model

et al. 2008

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“…Calculation of specific content of cytochrome P-450 in microsomes was based on an extinction coefficient of 91.5 nmol-' cm-', and cytochrome P-420 on an extinction coefficient of 110 nmol-l cm-' ~Monoclonal antibody designated MAb 1-12-3 to cytochrome P-450E was obtained as previously described (Parks et al 1986) in ascites fluid. This antibody was used in immunoblotting according to methods described by Kloepper-Sams et al (1987). Immunoblot analysis was carried out on liver mlcrosomes of Platichthys flesus that had been shipped on dry ice from Oslo to the Woods Hole Oceanographic Institution USA.…”

Section: Bakkementioning

confidence: 99%

Apparent cytochrome P-450 induction as an indication of exposure to environmental chemicals the flounder Platichthys flesus

Stegeman¹,

Woodin²,

Goksøyr³

1988

Mar. Ecol. Prog. Ser.

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“…Immunoblotting procedures used here were modified from those previously described (30). Briefly, microsomal proteins (10-80 ,..g/lane) were resolved on 12% SDS-PAGE gels and transferred to either nitrocellulose or nylon membrane.…”

Section: Methodsmentioning

confidence: 99%

“…These fish had appreciable levels ofCYP1A and higher levels of CYP2B-like protein than did the fish used in the treatment experiments. Normal rabbit IgG, the polyclonal antibody 118 against scup CYPlA (30), or the polyclonal antibody 7-94 against scup P450B (33) were added to the reaction mixtures at a protein/total P450 ratio of 4, 8, or 16 mg total IgG/nmol ofP450. After 15 min at 30°C, [1-14 C]AA was added, and the reaction was initiated with NADPH.…”

Section: Methodsmentioning

confidence: 99%

Involvement of Cytochrome P450 1A in the toxicity of aryl hydrocarbon receptor agonists : alteration arachidonic acid metabolism and production of reactive oxygen species

Schlezinger¹

1998

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Specificity and cross-reactivity of monoclonal and polyclonal antibodies against cytochrome P-450E of the marine fish scup

Cited by 132 publications

References 25 publications

Development of the morpholino gene knockdown technique in Fundulus heteroclitus: A tool for studying molecular mechanisms in an established environmental model

Development of the morpholino gene knockdown technique in Fundulus heteroclitus: A tool for studying molecular mechanisms in an established environmental model

Apparent cytochrome P-450 induction as an indication of exposure to environmental chemicals the flounder Platichthys flesus

Involvement of Cytochrome P450 1A in the toxicity of aryl hydrocarbon receptor agonists : alteration arachidonic acid metabolism and production of reactive oxygen species

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