Linked Life Data

21704603

Source:http://linkedlifedata.com/resource/pubmed/id/21704603

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
2011-7-25
pubmed:abstractText
Mapping protein interactions by immunoprecipitation is limited by the availability of antibodies recognizing available native epitopes within protein complexes with sufficient affinity. Here we demonstrate a scalable approach for generation of such antibodies using phage display and affinity maturation. We combined antibody variable heavy (V(H)) genes from target-specific clones (recognizing Src homology 2 (SH2) domains of LYN, VAV1, NCK1, ZAP70, PTPN11, CRK, LCK, and SHC1) with a repertoire of 10(8) to 10(9) new variable light (V(L)) genes. Improved binders were isolated by stringent selections from these new "chain-shuffled" libraries. We also developed a predictive 96-well immunocapture screen and found that only 12% of antibodies had sufficient affinity/epitope availability to capture endogenous target from lysates. Using antibodies of different affinities to the same epitope, we show that affinity improvement was a key determinant for success and identified a clear affinity threshold value (60 nM for SHC1) that must be breached for success in immunoprecipitation. By combining affinity capture using matured antibodies to SHC1 with mass spectrometry, we identified seven known binding partners and two known SHC1 phosphorylation sites in epidermal growth factor (EGF)-stimulated human breast cancer epithelial cells. These results demonstrate that antibodies capable of immunoprecipitation can be generated by chain shuffling, providing a scalable approach to mapping protein-protein interaction networks.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Oct
pubmed:issn
1096-0309
pubmed:author
pubmed:copyrightInfo
Copyright © 2011 Elsevier Inc. All rights reserved.
pubmed:issnType
Electronic
pubmed:day
1
pubmed:volume
417
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
25-35
pubmed:meshHeading
pubmed:year
2011
pubmed:articleTitle
Mapping protein interactions by combining antibody affinity maturation and mass spectrometry.
pubmed:affiliation
Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, UK.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't, Research Support, N.I.H., Extramural