Antibodies immobilized as arrays to profile protein post-translational modifications in mammalian cells.
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.
Ivanov
Stanimir S
SS
Department of Pathology & Laboratory Medicine, Brown University Medical School, Providence, RI 02912, USA.
Chung
Alicia S
AS
Yuan
Zheng-long
ZL
Guan
Ying-jie
YJ
Sachs
Katherine V
KV
Reichner
Jonathan S
JS
Chin
Y Eugene
YE
D016475:3T3 Cells; D000107:Acetylation; D000818:Animals; D000906:Antibodies; D002460:Cell Line; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D020869:Gene Expression Profiling; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D010766:Phosphorylation; D040081:Protein Array Analysis; D011499:Protein Processing, Post-Translational; D014443:Tyrosine / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*
D000906:Antibodies; D005456:Fluorescent Dyes; D025801:Ubiquitin; D014443:Tyrosine