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DATASHEET Description: Endothelins are a family of peptides that are potent vasoconstrictors produced by mammalian vascular endothelial cells. The receptors for endothelins are widely expressed on tissues. This sheep antibody recognizes epitopes on the cytoplasmic region of the ET(A) Receptor. The binding of this antibody does not interfere with the function of the receptor so that endothelins can interact with the receptor while the antibody is present. Storage: Aliquot and store at -20° C or colder. once thawed store at 4° C with the addition of 0.1 % sodium azide or other preservative. Avoid repeated freeze-thaw cycles. Technical Information: This antibody recognizes epitopes on the cytoplasmic side of the receptor and will not block the binding of endothelins. Therefore, the cell membrane must be permeable to the antibody. IMMUNOCYTOCHEMICAL STAINING FOR ET(A) AND ET(B) RECEPTORS: Fixed paraffin-embedded sections (4-6 mm) are deparaffinized, stained, counterstained, dehydrated, cleared and mounted. Block the sections with 10 % normal rabbit serum to inhibit non-specific binding. Dilute the Protein G purified sheep anti-ET(A) (P00364) or anti-ET(B) (P00366) to 10 ug IgG/mL in PBS containing 1 % normal rabbit serum. Perform the immunoreactivity step by using a peroxidase-coupled rabbit anti-sheep IgG. IMMUNOPRECIPITATION OF ET(A) AND ET(B) RECEPTORS Prepare cell lysates (~5 mg) by homogenizing tissues in 2 ml RIPA (50 mM Tris, pH=7.5, 150 mM NACl, 1 % NP-40, 0.5 % deoxycholic acid, 0.1% SDS, 2 mM EDTA, 10 mM NaF, 10 ug/ml leupeptin, 10 ug/ml pepstatin, 20 ug/ml aprotinin). Centrifuge for 10 minutes at 1000 x g. Dilute 500 ul of the cell lysate 1:1 in RIPA buffer. Immunoprecipitate the receptors with 4 ug of antibody for 1 hour at room temperature. Collect the precipitin with Protein G Plus Agarose and wash 3 times with 1.0 ml RIPA buffer. Suspend the final pellet in 40 ul 2X SDS loading buffer, heated at 100° C for 5 minutes and recentrifuge for 5 minutes in a microcentrifuge. Load 40 ul of supernatant onto a 8-16 % gradient SDS-PAGE minigel. |
For Research Purposes Only
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Citations and Applications |
Wulfing P, Diallo R, Kersting C, Wulfing C, Poremba C, Greb RR, Bocker W, Kiesel L. Endothelin-1, Endothelin-A- and Endothelin-B-receptor expression in preinvasive and invasive breast disease. Oncol Rep. 2004 Apr;11(4):791-6. Abstract IH |
Siren AL, Knerlich F, Schilling L, Kamrowski-Kruck H, Hahn A, Ehrenreich H. Differential glial and vascular expression of endothelins and their receptors in rat brain after neurotrauma. Neurochem Res. 2000 Jul;25(7):957-69. Abstract IH |
Shraga-Levine Z, Sokolovsky M. Functional coupling of G proteins to endothelin receptors is ligand and receptor subtype specific. Cell Mol Neurobiol. 2000 Jun;20(3):305-17. Abstract IP |
Fernandes LB, Henry PJ, Spalding LJ, Cody SH, Pudney CJ, Goldie RG. Immunocytochemical detection of endothelin receptors in rat cultured airway nerves. J Cardiovasc Pharmacol. 1998;31 Suppl 1:S222-4. Abstract |
Barber DA, Michener SR, Ziesmer SC, Miller VM. Chronic increases in blood flow upregulate endothelin-B receptors in arterial smooth muscle. Am J Physiol. 1996 Jan;270(1 Pt 2):H65-71 Abstract |