DATASHEET

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.

IMMUNOCYTOCHEMIAL 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 1.0 % 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 the supernatant onto a 8-16 % gradient SDA-PAGE minigel.

For Research Purposes Only