HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: M800171-MCP200v1 8/2/245    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Glossary Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Wu, W. Articles by Lund-Johansen, F. Search for Related Content PubMed PubMed Citation Articles by Wu, W. Articles by Lund-Johansen, F. Social Bookmarking                      What's this?

Molecular & Cellular Proteomics 8:245-257, 2009.
© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

---

Research

Antibody Array Analysis with Label-based Detection and Resolution of Protein Size ^*,S

Weiwei Wu, Heidi Slåstad^,, Daniel de la Rosa Carrillo^,¶, Tom Frey^||, Geir Tjønnfjord, Eva Boretti, Hans-Christian Aasheim^**, Vaclav Horejsi and Fridtjof Lund-Johansen^,,

From the Rikshospitalet Medical Center and the University of Oslo, Department of Immunology, Department of Medicine, Section for Hematology, and ^¶ Department of Dermatology, 0027 Oslo, Norway, ^|| BD Biosciences, San Jose, California 95131, Department of Medical Genetics, ^** Ullevaal University Hospital, 0407 Oslo, Norway, and Academy of Sciences of the Czech Republic, Institute of Molecular Genetics, Videnska 1083, CA-142 20, Prague 4Krc, Czech Republic

Elements from DNA microarray analysis, such as sample labeling and micro-spotting of capture reagents, have been successfully adapted to multiplex measurements of soluble cytokines. Application in cell biology is hampered by the lack of mono-specific antibodies and the fact that many proteins occur in complexes. Here, we incorporated a principle from Western blotting and resolved protein size as an additional parameter. Proteins from different cellular compartments were labeled and separated by size exclusion chromatography into 20 fractions. All were analyzed with replicate antibody arrays. The elution profiles of all antibody targets were compiled to color maps that resemble Western blots with bands of antibody reactivity across the size separation range (670–10 kDa). A new solid phase designed for processing in microwell plates was developed to handle the large number of samples. Antibodies were bound to protein G-coupled microspheres surface-labeled with 300 combinations of four fluorescent dyes. Fluorescence from particle color codes and the protein label were measured by high-speed flow cytometry. Cytoplasmic protein kinases were detected as bands near predictable elution points. For proteins with atypical elution characteristics or multiple contexts, two or more antibodies were used as internal references of specificity. Membrane proteins eluted near the void volume, and additional bands corresponding to intracellular forms were detected for several targets. Elution profiles of cyclin-dependent kinases (cdks), cyclins, and cyclin-dependent kinase inhibitors, were compatible with their occurrence in complexes that vary with the cell cycle phase and subcellular localization. A two-dimensional platform circumvents the need for mono-specific capture antibodies and extends the utility of antibody array analysis to studies of protein complexes.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?