Advanced Search

Journal Navigation

Journal Home

Subscriptions

Archive

Contact Us

Table of Contents

Click here for more information

Sign In to gain access to subscriptions and/or personal tools.
Journal of Biomolecular Screening
This Article
Right arrow Full Text (PDF)
Right arrow References
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to Saved Citations
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Request Reprints
Right arrow Add to My Marked Citations
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (17)
Right arrow Citing Articles via Google Scholar
Right arrow Citing Articles via Scopus
Google Scholar
Right arrow Articles by Wu, J. J.
Right arrow Articles by Sills, M. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wu, J. J.
Right arrow Articles by Sills, M. A.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Identification of a High-Affinity Anti-Phosphoserine Antibody for the Development of a Homogeneous Fluorescence Polarization Assay of Protein Kinase C

Jinzi J. Wu

Immunex Corporation, Seattle, WA

Donna R. Yarwood

Novartis Institute for Biomedical Research, Novartis Pharmaceuticals Corporation, Summit, NJ

Quynhchi Pham

Novartis Institute for Biomedical Research, Novartis Pharmaceuticals Corporation, Summit, NJ

Matthew A. Sills

Novartis Institute for Biomedical Research, Novartis Pharmaceuticals Corporation, Summit, NJ

In the last few years, fluorescence polarization (FP) has been applied to the development of robust, homogeneous, high throughput assays in molecular recognition research, such as ligand-protein interactions. Recently, this technology has been applied to the development of homogeneous tyrosine kinase assays, since there are high-affinity anti-phosphotyrosine antibodies available. Unlike tyrosine kinases, application of FP to assay development for serine/threonine kinases has been impeded because of lack of high-affinity anti-phosphoserine/threonine antibodies. In the present study, we report the discovery of a high-affinity, monoclonal anti-phosphoserine antibody, 2B9, with a Kd of 250 ± 34 pM for a phosphoserine-containing peptide tracer, fluorescein-RFARKGS(PO4)LRQKNV. Our data suggest that 2B9 is selective for fluorescein-RFARKGS(PO4)LRQKNV. The antibody and tracer have been used for the development of a competitive FP assay for protein kinase C (PKC) in 384-well plates. Phosphatidylserine, which enhances the kinase activity of PKC in a Ca2+-dependent manner and has a structure similar to that of phosphoserine, did not interfere with binding of the peptide tracer to the antibody in the FP assay. The data indicate that the FP assay is more sensitive and robust than the scintillation proximity assay for PKC. The FP assay developed here can be used for rapid screening of hundreds of thousands of compounds for discovery of therapeutic leads for PKC-related diseases.

Journal of Biomolecular Screening, Vol. 5, No. 1, 23-30 (2000)
DOI: 10.1177/108705710000500106
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?


This article has been cited by other articles:


Home page
 J Biomol ScreenHome page
Y. Du, S. C. Masters, F. R. Khuri, and H. Fu
Monitoring 14-3-3 Protein Interactions with a Homogeneous Fluorescence Polarization Assay
J Biomol Screen, April 1, 2006; 11(3): 269 - 276.
[Abstract] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
F. Rininsland, W. Xia, S. Wittenburg, X. Shi, C. Stankewicz, K. Achyuthan, D. McBranch, and D. Whitten
Metal ion-mediated polymer superquenching for highly sensitive detection of kinase and phosphatase activities
PNAS, October 26, 2004; 101(43): 15295 - 15300.
[Abstract] [Full Text] [PDF]

Home page
 J Biomol ScreenHome page
T. C. Turek-Etienne, M. Lei, J. S. Terracciano, E. F. Langsdorf, R. W. Bryant, R. F. Hart, and A. C. Horan
Use of Red-Shifted Dyes in a Fluorescence Polarization AKT Kinase Assay for Detection of Biological Activity in Natural Product Extracts
J Biomol Screen, February 1, 2004; 9(1): 52 - 61.
[Abstract] [PDF]

Home page
 J Biomol ScreenHome page
E. A. Gaudet, K.-S. Huang, Y. Zhang, W. Huang, D. Mark, and J. R. Sportsman
A Homogeneous Fluorescence Polarization Assay Adaptable for a Range of Protein Serine/Threonine and Tyrosine Kinases
J Biomol Screen, April 1, 2003; 8(2): 164 - 175.
[Abstract] [PDF]

Home page
 J Biomol ScreenHome page
J. C. Owicki
Fluorescence Polarization and Anisotropy in High Throughput Screening: Perspectives and Primer
J Biomol Screen, October 1, 2000; 5(5): 297 - 306.
[Abstract] [PDF]