Development of a sensitive peptide-based immunoassay: Application to detection of the Jun and Fos oncoproteins. Heuer, K., Mackay, J., Podzebenko, P., Bains, N., Weiss, A., King, G., & Easterbrook-Smith, S. Biochemistry, 35(28):9069-9075, 1996. doi abstract bibtex c-Jun and c-Fos belong to the bZIP class of transcriptional activator proteins, many of which have been implicated in the neoplastic transformation of cells. We are interested in engineering dominant-negative leucine zipper (LZ) peptides as a means of sequestering these proteins in vivo in order to suppress their transcriptional regulatory activity. Toward this end, we have developed a novel immunoassay for measuring the dimerization affinities of dimeric Jun and Fos complexes. This peptide-based ELISA relies on the fact that Jun and Fos preferentially form heterodimers via their leucine zipper domains. Recombinant Jun leucine zipper peptides (either native JunLZ or a V36→E point mutant) were labeled with biotin and specifically bound through a leucine zipper interaction to a FosLZ-glutathione S-transferase fusion protein adsorbed onto the wells of an ELISA tray. Jun:Fos complexes were subsequently detected using a recently developed streptavidin-based amplification system known as enzyme complex amplification [Wilson, M. R., and Easterbrook-Smith, S. B. (1993) Anal. Biochem. 209, 183-187]. This ELISA system can detect subnanomolar concentrations of Jun and Fos, thus allowing determination of the dissociation constants for complex formation. The dissociation constant for formation of the native JunLZ:FosLZ heterodimer at 37 °C was determined to be 0.99 ± 0.30 nM, while that for JunLZ(V36E):FosLZ heterodimer was 0.90 ± 0.13 μM. These results demonstrate that the novel peptide-based ELISA described herein is simple and sensitive and can be used to rapidly screen for potential dominant-negative leucine zipper peptides.
@article{
title = {Development of a sensitive peptide-based immunoassay: Application to detection of the Jun and Fos oncoproteins},
type = {article},
year = {1996},
pages = {9069-9075},
volume = {35},
id = {3ec35428-2577-35f5-8d93-7aae09b3b3bc},
created = {2023-01-10T01:46:59.013Z},
file_attached = {false},
profile_id = {a5a2ab6f-a8b5-3db6-97bc-618752ee4386},
group_id = {bc1ab1d4-9e57-37e6-9fb5-435fca0ee9d2},
last_modified = {2023-01-10T01:46:59.013Z},
read = {false},
starred = {false},
authored = {false},
confirmed = {false},
hidden = {false},
private_publication = {false},
abstract = {c-Jun and c-Fos belong to the bZIP class of transcriptional activator proteins, many of which have been implicated in the neoplastic transformation of cells. We are interested in engineering dominant-negative leucine zipper (LZ) peptides as a means of sequestering these proteins in vivo in order to suppress their transcriptional regulatory activity. Toward this end, we have developed a novel immunoassay for measuring the dimerization affinities of dimeric Jun and Fos complexes. This peptide-based ELISA relies on the fact that Jun and Fos preferentially form heterodimers via their leucine zipper domains. Recombinant Jun leucine zipper peptides (either native JunLZ or a V36→E point mutant) were labeled with biotin and specifically bound through a leucine zipper interaction to a FosLZ-glutathione S-transferase fusion protein adsorbed onto the wells of an ELISA tray. Jun:Fos complexes were subsequently detected using a recently developed streptavidin-based amplification system known as enzyme complex amplification [Wilson, M. R., and Easterbrook-Smith, S. B. (1993) Anal. Biochem. 209, 183-187]. This ELISA system can detect subnanomolar concentrations of Jun and Fos, thus allowing determination of the dissociation constants for complex formation. The dissociation constant for formation of the native JunLZ:FosLZ heterodimer at 37 °C was determined to be 0.99 ± 0.30 nM, while that for JunLZ(V36E):FosLZ heterodimer was 0.90 ± 0.13 μM. These results demonstrate that the novel peptide-based ELISA described herein is simple and sensitive and can be used to rapidly screen for potential dominant-negative leucine zipper peptides.},
bibtype = {article},
author = {Heuer, K.H. and Mackay, J.P. and Podzebenko, P. and Bains, N.P.S. and Weiss, A.S. and King, G.F. and Easterbrook-Smith, S.B.},
doi = {10.1021/bi952817o},
journal = {Biochemistry},
number = {28}
}
Downloads: 0
{"_id":"BjHxXw4AqrBBofswc","bibbaseid":"heuer-mackay-podzebenko-bains-weiss-king-easterbrooksmith-developmentofasensitivepeptidebasedimmunoassayapplicationtodetectionofthejunandfosoncoproteins-1996","authorIDs":["ZTncL5PqZZ6RafCsL"],"author_short":["Heuer, K.","Mackay, J.","Podzebenko, P.","Bains, N.","Weiss, A.","King, G.","Easterbrook-Smith, S."],"bibdata":{"title":"Development of a sensitive peptide-based immunoassay: Application to detection of the Jun and Fos oncoproteins","type":"article","year":"1996","pages":"9069-9075","volume":"35","id":"3ec35428-2577-35f5-8d93-7aae09b3b3bc","created":"2023-01-10T01:46:59.013Z","file_attached":false,"profile_id":"a5a2ab6f-a8b5-3db6-97bc-618752ee4386","group_id":"bc1ab1d4-9e57-37e6-9fb5-435fca0ee9d2","last_modified":"2023-01-10T01:46:59.013Z","read":false,"starred":false,"authored":false,"confirmed":false,"hidden":false,"private_publication":false,"abstract":"c-Jun and c-Fos belong to the bZIP class of transcriptional activator proteins, many of which have been implicated in the neoplastic transformation of cells. We are interested in engineering dominant-negative leucine zipper (LZ) peptides as a means of sequestering these proteins in vivo in order to suppress their transcriptional regulatory activity. Toward this end, we have developed a novel immunoassay for measuring the dimerization affinities of dimeric Jun and Fos complexes. This peptide-based ELISA relies on the fact that Jun and Fos preferentially form heterodimers via their leucine zipper domains. Recombinant Jun leucine zipper peptides (either native JunLZ or a V36→E point mutant) were labeled with biotin and specifically bound through a leucine zipper interaction to a FosLZ-glutathione S-transferase fusion protein adsorbed onto the wells of an ELISA tray. Jun:Fos complexes were subsequently detected using a recently developed streptavidin-based amplification system known as enzyme complex amplification [Wilson, M. R., and Easterbrook-Smith, S. B. (1993) Anal. Biochem. 209, 183-187]. This ELISA system can detect subnanomolar concentrations of Jun and Fos, thus allowing determination of the dissociation constants for complex formation. The dissociation constant for formation of the native JunLZ:FosLZ heterodimer at 37 °C was determined to be 0.99 ± 0.30 nM, while that for JunLZ(V36E):FosLZ heterodimer was 0.90 ± 0.13 μM. These results demonstrate that the novel peptide-based ELISA described herein is simple and sensitive and can be used to rapidly screen for potential dominant-negative leucine zipper peptides.","bibtype":"article","author":"Heuer, K.H. and Mackay, J.P. and Podzebenko, P. and Bains, N.P.S. and Weiss, A.S. and King, G.F. and Easterbrook-Smith, S.B.","doi":"10.1021/bi952817o","journal":"Biochemistry","number":"28","bibtex":"@article{\n title = {Development of a sensitive peptide-based immunoassay: Application to detection of the Jun and Fos oncoproteins},\n type = {article},\n year = {1996},\n pages = {9069-9075},\n volume = {35},\n id = {3ec35428-2577-35f5-8d93-7aae09b3b3bc},\n created = {2023-01-10T01:46:59.013Z},\n file_attached = {false},\n profile_id = {a5a2ab6f-a8b5-3db6-97bc-618752ee4386},\n group_id = {bc1ab1d4-9e57-37e6-9fb5-435fca0ee9d2},\n last_modified = {2023-01-10T01:46:59.013Z},\n read = {false},\n starred = {false},\n authored = {false},\n confirmed = {false},\n hidden = {false},\n private_publication = {false},\n abstract = {c-Jun and c-Fos belong to the bZIP class of transcriptional activator proteins, many of which have been implicated in the neoplastic transformation of cells. We are interested in engineering dominant-negative leucine zipper (LZ) peptides as a means of sequestering these proteins in vivo in order to suppress their transcriptional regulatory activity. Toward this end, we have developed a novel immunoassay for measuring the dimerization affinities of dimeric Jun and Fos complexes. This peptide-based ELISA relies on the fact that Jun and Fos preferentially form heterodimers via their leucine zipper domains. Recombinant Jun leucine zipper peptides (either native JunLZ or a V36→E point mutant) were labeled with biotin and specifically bound through a leucine zipper interaction to a FosLZ-glutathione S-transferase fusion protein adsorbed onto the wells of an ELISA tray. Jun:Fos complexes were subsequently detected using a recently developed streptavidin-based amplification system known as enzyme complex amplification [Wilson, M. R., and Easterbrook-Smith, S. B. (1993) Anal. Biochem. 209, 183-187]. This ELISA system can detect subnanomolar concentrations of Jun and Fos, thus allowing determination of the dissociation constants for complex formation. The dissociation constant for formation of the native JunLZ:FosLZ heterodimer at 37 °C was determined to be 0.99 ± 0.30 nM, while that for JunLZ(V36E):FosLZ heterodimer was 0.90 ± 0.13 μM. These results demonstrate that the novel peptide-based ELISA described herein is simple and sensitive and can be used to rapidly screen for potential dominant-negative leucine zipper peptides.},\n bibtype = {article},\n author = {Heuer, K.H. and Mackay, J.P. and Podzebenko, P. and Bains, N.P.S. and Weiss, A.S. and King, G.F. and Easterbrook-Smith, S.B.},\n doi = {10.1021/bi952817o},\n journal = {Biochemistry},\n number = {28}\n}","author_short":["Heuer, K.","Mackay, J.","Podzebenko, P.","Bains, N.","Weiss, A.","King, G.","Easterbrook-Smith, S."],"biburl":"https://bibbase.org/service/mendeley/a5a2ab6f-a8b5-3db6-97bc-618752ee4386","bibbaseid":"heuer-mackay-podzebenko-bains-weiss-king-easterbrooksmith-developmentofasensitivepeptidebasedimmunoassayapplicationtodetectionofthejunandfosoncoproteins-1996","role":"author","urls":{},"metadata":{"authorlinks":{"mackay, j":"https://bibbase.org/service/mendeley/a5a2ab6f-a8b5-3db6-97bc-618752ee4386/group/bc1ab1d4-9e57-37e6-9fb5-435fca0ee9d2"}},"downloads":0},"bibtype":"article","creationDate":"2020-08-19T06:33:00.731Z","downloads":0,"keywords":[],"search_terms":["development","sensitive","peptide","based","immunoassay","application","detection","jun","fos","oncoproteins","heuer","mackay","podzebenko","bains","weiss","king","easterbrook-smith"],"title":"Development of a sensitive peptide-based immunoassay: Application to detection of the Jun and Fos oncoproteins","year":1996,"biburl":"https://bibbase.org/service/mendeley/a5a2ab6f-a8b5-3db6-97bc-618752ee4386","dataSources":["QNo3CKhtog6c3sh7Y","ya2CyA73rpZseyrZ8","4BKYoCQJ3ndMcbbtH","2252seNhipfTmjEBQ","ibLPPAndxdcRpxcwH"]}