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Probing Human beta1- And beta2 -Adrenoceptors With Domain-Specific Fusion Protein Antibodies

Probing Human beta1- And beta2 -Adrenoceptors With Domain-Specific Fusion Protein Antibodies

R Jahns, C Siegmund, V Jahns, H Reiländer, A Maidhof, W Müller-Esterl, M J Lohse, F Boege

Eur J Pharmacol. 1997 Sep 3;334(1):115-26.

PMID: 9346338

Abstract:

In order to generate antibodies suitable for immunological studies on beta-adrenoceptors constitutively expressed at low levels in cells or tissues we have produced fusion proteins of the amino- and carboxy-terminus, and the second extracellular loop of the human beta1- or beta2-adrenoceptors with bacterial glutathione-S-transferase in E. coli. Rabbit antibodies raised against these fusion proteins strongly reacted with intact human beta1- or beta2-adrenoceptors in a subtype- and domain-specific manner. Antibodies directed against the second extracellular loop of the beta1-adrenoceptor reacted stronger with non-denatured receptors and decreased the affinity of the 3H-labelled antagonist (-)-4-(3-t-butylamino-2-hydroxypropoxy)-[5,7-3H]benzimidazol-2-one ([3H]CGP 12 177), indicating a specific interaction with the native receptor. In contrast, antibodies directed against carboxy- and amino-terminal receptor domains reacted strongly both with denatured and non-denatured receptors but did not interfere with binding of [3H]CGP 12 177. Affinity purified antibodies were used for detecting the beta1- or the beta2-adrenoceptor subtype heterologously produced in Sf9 cells by enzyme-linked immunosorbent assay, Western blotting, immunoprecipitation, and indirect immunofluorescence microscopy. Moreover, we could demonstrate that avidity, titers, and specificity of these antibodies were high enough for studying beta-adrenoceptors constitutively expressed in human A431 cells, where we observed a patched membrane distribution of the receptors.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
IAR42416599	Anti-phospho-IKK-α/β (pSer176/177) antibody produced in rabbit			Price

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