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WeightNameValue
1000 Titel
  • Completion of Proteomic Data Sets by Kd Measurement Using Cell-Free Synthesis of Site-Specifically Labeled Proteins
1000 Autor/in
  1. Majkut, Paul |
  2. Claußnitzer, Iris |
  3. Merk, Helmut |
  4. Freund, Christian |
  5. Gerrits, Michael |
  6. Hackenberger, Christian |
1000 Erscheinungsjahr 2013
1000 LeibnizOpen
1000 Publikationstyp
  1. Artikel |
1000 Online veröffentlicht
  • 2013-12-10
1000 Erschienen in
1000 Quellenangabe
  • 8(12): e82352
1000 FRL-Sammlung
1000 Copyrightjahr
  • 2013
1000 Lizenz
1000 Verlagsversion
  • https://doi.org/10.1371/journal.pone.0082352 |
  • https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3858276/ |
1000 Ergänzendes Material
  • http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0082352# |
1000 Publikationsstatus
1000 Begutachtungsstatus
1000 Sprache der Publikation
1000 Abstract/Summary
  • The characterization of phosphotyrosine mediated protein-protein interactions is vital for the interpretation of downstream pathways of transmembrane signaling processes. Currently however, there is a gap between the initial identification and characterization of cellular binding events by proteomic methods and the in vitro generation of quantitative binding information in the form of equilibrium rate constants (Kd values). In this work we present a systematic, accelerated and simplified approach to fill this gap: using cell-free protein synthesis with site-specific labeling for pull-down and microscale thermophoresis (MST) we were able to validate interactions and to establish a binding hierarchy based on Kd values as a completion of existing proteomic data sets. As a model system we analyzed SH2-mediated interactions of the human T-cell phosphoprotein ADAP. Putative SH2 domain-containing binding partners were synthesized from a cDNA library using Expression-PCR with site-specific biotinylation in order to analyze their interaction with fluorescently labeled and in vitro phosphorylated ADAP by pull-down. On the basis of the pull-down results, selected SH2’s were subjected to MST to determine Kd values. In particular, we could identify an unexpectedly strong binding of ADAP to the previously found binding partner Rasa1 of about 100 nM, while no evidence of interaction was found for the also predicted SH2D1A. Moreover, Kd values between ADAP and its known binding partners SLP-76 and Fyn were determined. Next to expanding data on ADAP suggesting promising candidates for further analysis in vivo, this work marks the first Kd values for phosphotyrosine/SH2 interactions on a phosphoprotein level.
1000 Sacherschließung
lokal SH2 domain
lokal Proteomic databases
lokal Phosphorylation
lokal Protein-protein interactions
lokal Protein interactions
lokal Polymerase chain reaction
lokal Protein interaction networks
lokal Sequence motif analysis
1000 Fächerklassifikation (DDC)
1000 Liste der Beteiligten
  1. https://frl.publisso.de/adhoc/creator/TWFqa3V0LCBQYXVsIA==|https://frl.publisso.de/adhoc/creator/Q2xhdcOfbml0emVyLCBJcmlzIA==|https://frl.publisso.de/adhoc/creator/TWVyaywgSGVsbXV0IA==|https://frl.publisso.de/adhoc/creator/RnJldW5kLCBDaHJpc3RpYW4g|https://frl.publisso.de/adhoc/creator/R2Vycml0cywgTWljaGFlbCA=|http://orcid.org/0000-0001-7457-4742
1000 Label
1000 Förderer
  1. Boehringer Ingelheim Stiftung |
  2. Deutsche Forschungsgesellschaft (Sonderforschungsbereich 765) |
  3. Fonds der Chemischen Industrie |
  4. Federal Ministry of Education and Research |
1000 Fördernummer
  1. -
  2. -
  3. -
  4. 0315027A.
1000 Förderprogramm
  1. Plus 3 program
  2. -
  3. -
  4. -
1000 Dateien
1000 Förderung
  1. 1000 joinedFunding-child
    1000 Förderer Boehringer Ingelheim Stiftung |
    1000 Förderprogramm Plus 3 program
    1000 Fördernummer -
  2. 1000 joinedFunding-child
    1000 Förderer Deutsche Forschungsgesellschaft (Sonderforschungsbereich 765) |
    1000 Förderprogramm -
    1000 Fördernummer -
  3. 1000 joinedFunding-child
    1000 Förderer Fonds der Chemischen Industrie |
    1000 Förderprogramm -
    1000 Fördernummer -
  4. 1000 joinedFunding-child
    1000 Förderer Federal Ministry of Education and Research |
    1000 Förderprogramm -
    1000 Fördernummer 0315027A.
1000 Objektart article
1000 Beschrieben durch
1000 @id frl:6402871.rdf
1000 Erstellt am 2017-06-07T09:56:00.956+0200
1000 Erstellt von 23
1000 beschreibt frl:6402871
1000 Bearbeitet von 218
1000 Zuletzt bearbeitet 2022-08-18T08:01:26.653+0200
1000 Objekt bearb. Mon Dec 07 16:49:35 CET 2020
1000 Vgl. frl:6402871
1000 Oai Id
  1. oai:frl.publisso.de:frl:6402871 |
1000 Sichtbarkeit Metadaten public
1000 Sichtbarkeit Daten public
1000 Gegenstand von

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