Antibody array analysis with label-based detection and resolution of protein size

Area of Science:

• Proteomics
• Molecular Biology
• Biochemistry

Background:

• Multiplex measurements of soluble cytokines using DNA microarray principles are established.
• Cell biology studies are limited by the lack of mono-specific antibodies and the prevalence of protein complexes.

Purpose of the Study:

• To develop a novel two-dimensional antibody array platform for analyzing protein complexes.
• To overcome the limitations of mono-specific antibody requirements in traditional antibody array analysis.

Main Methods:

• Proteins were labeled and separated by size exclusion chromatography into 20 fractions.
• Antibody arrays were used with protein G-coupled microspheres and fluorescent dyes for high-speed flow cytometry analysis.
• A new solid phase for microwell plates was developed for high-throughput sample processing.

Main Results:

• A two-dimensional color map resembling Western blots was generated, showing antibody reactivity across a size separation range (670-10 kDa).
• Cytoplasmic protein kinases, membrane proteins, and intracellular forms were detected at predictable elution points.
• Elution profiles of cyclin-dependent kinases (cdks), cyclins, and inhibitors indicated their presence in cell cycle- and localization-dependent complexes.

Conclusions:

• The developed two-dimensional platform circumvents the need for mono-specific capture antibodies.
• This approach extends the utility of antibody array analysis to the study of protein complexes and their dynamics.