%0 Journal Article %1 Jahns1996 %A Jahns, R. %A Siegmund, C. %A Jahns, V. %A Reilander, H. %A Maidhof, A. %A Muller-Esterl, W. %A Lohse, M. J. %A Boege, F. %D 1996 %J Eur J Pharmacol %K *Antibodies Animals Antibody Assay Fluorescence Fusion Humans Microscopy, Precipitin Protein Proteins/*immunology Rabbits Radioligand Recombinant Secondary Specificity Structure, Tests beta-1/*analysis beta-2/*analysis Receptor Adrenergic %N 1 %P 111-21 %T Probing human beta 1- and beta 2-adrenoceptors with domain-specific fusion protein antibodies %U http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=8982658 %V 316 %X 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 beta 1- or beta 2-adrenoceptors with bacterial glutathione-S-transferase in E. coli. Rabbit antibodies raised against these fusion proteins strongly reacted with intact human beta 1- or beta 2-adrenoceptors in a subtype- and domain-specific manner. Antibodies directed against the second extracellular loop of the beta 1-adrenoceptor reacted stronger with non-denatured receptors and decreased the affinity of the 3H-labelled antagonist (-)-4-(3-t-butylamino-2-hydroxypropoxy)-5,7-3Hbenzimidazol-2-one (3HCGP 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 3HCGP 12 177. Affinity purified antibodies were used for detecting the beta 1- or the beta 2-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.

@article{Jahns1996, 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 beta 1- or beta 2-adrenoceptors with bacterial glutathione-S-transferase in E. coli. Rabbit antibodies raised against these fusion proteins strongly reacted with intact human beta 1- or beta 2-adrenoceptors in a subtype- and domain-specific manner. Antibodies directed against the second extracellular loop of the beta 1-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 beta 1- or the beta 2-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.}, added-at = {2010-12-14T18:12:02.000+0100}, author = {Jahns, R. and Siegmund, C. and Jahns, V. and Reilander, H. and Maidhof, A. and Muller-Esterl, W. and Lohse, M. J. and Boege, F.}, biburl = {https://www.bibsonomy.org/bibtex/2ac0b3aa2953ac877a3d5504ce05c714f/pharmawuerz}, endnotereftype = {Journal Article}, interhash = {19e92ff91684d0bb00fe4069e0262e98}, intrahash = {ac0b3aa2953ac877a3d5504ce05c714f}, issn = {0014-2999 (Print) 0014-2999 (Linking)}, journal = {Eur J Pharmacol}, keywords = {*Antibodies Animals Antibody Assay Fluorescence Fusion Humans Microscopy, Precipitin Protein Proteins/*immunology Rabbits Radioligand Recombinant Secondary Specificity Structure, Tests beta-1/*analysis beta-2/*analysis Receptor Adrenergic}, month = {Nov 28}, note = {Jahns, R Siegmund, C Jahns, V Reilander, H Maidhof, A Muller-Esterl, W Lohse, M J Boege, F Netherlands European journal of pharmacology Eur J Pharmacol. 1996 Nov 28;316(1):111-21.}, number = 1, pages = {111-21}, shorttitle = {Probing human beta 1- and beta 2-adrenoceptors with domain-specific fusion protein antibodies}, timestamp = {2010-12-14T18:22:40.000+0100}, title = {Probing human beta 1- and beta 2-adrenoceptors with domain-specific fusion protein antibodies}, url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=8982658}, volume = 316, year = 1996 }