Antibodies Immobilized as Arrays to Profile Protein Post-translational Modifications in Mammalian Cells

Ying-jie Guan,Alicia S. Chung,Jonathan S. Reichner,Zheng-long Yuan,Katherine V. Sachs,Stanimir S. Ivanov,Y. Eugene Chin

doi:10.1074/mcp.m300130-mcp200

Abstract

Previously, we demonstrated that antibodies printed on a solid support were able to detect protein-protein interaction in mammalian cells. Here we further developed the antibody array system for detecting proteins with various post-translational modifications in mammalian cells. In this novel approach, immunoprecipitated proteins were labeled with fluorescent dye followed by incubation over antibody arrays. Targeted proteins, captured by the antibodies immobilized on PVDF membrane or glass slide, were detected by means of near infrared fluorescent scanner or fluorescent microscopy. To demonstrate the application of the antibody arrays in protein post-translational modifications, we profiled protein tyrosine phosphorylation, ubiquitination, and acetylation in mammalian cells under different conditions. Our results indicate that antibody array technology can provide a powerful means of profiling a large number of proteins with different post-translational modifications in cells.

Highlights

We demonstrated that antibodies printed on a solid support were able to detect protein-protein interaction in mammalian cells
Caspase1 and IRF-1 proteins were detected in STAT1ϩ/ϩ but not in STAT1Ϫ/Ϫ mouse fibroblast cells (Fig. 1a), in accordance with the previous findings that STAT1 plays an essential role in the expression of these two genes (9 –11)
Cy2- and Cy5-labeled recombinant His6STAT1 proteins (Fig. 1b) were mixed at different ratios and incubated with a glass slide immobilized with anti-His6 antibody, anti-STAT1 antibody, or control immunoglobulin G (IgG)

Summary

Introduction

We demonstrated that antibodies printed on a solid support were able to detect protein-protein interaction in mammalian cells. Results demonstrate an expanded application of the use of antibody arrays for profiling proteins with alterations in tyrosine phosphorylation, ubiquitination, and acetylation in mammalian cells in response to various treatments.

Results

Conclusion