( Qian Gao Kris Simonyi...)Detection of Phosphorylated and Total ERK and p38 MAPK by Bio-Plex Assay, ELISA, and Western Blotting, Rev A

Qian Gao, Kris Simonyi, and Claudia Suen, Bio-Rad Laboratories, Inc., 2000 Alfred Nobel Drive, Hercules, CA 94547 USA

Introduction
Western blotting is a traditional technique for detecting phosphorylated proteins within a variety of cell culture and tissue sample lysates. In recent years, new techniques that apply a sandwich antibody capture approach have become available for detecting phosphorylated proteins. These techniques include Bio-Plex phosphoprotein assays, which are multiplex bead-based assays for the detection of one or more different phosphorylated proteins within a single sample. Enzyme-linked immunosorbent assays (ELISA) are another technique that has been increasingly applied to detect phosphorylated proteins a single target at a time. In this study, commercially available Bio-Plex phosphoprotein and total target assays, and phosphoprotein and total ELISA assays, are compared with western blotting in terms of sensitivity, detection range, and level of correlation, for the detection of phospho- and total ERK and p38 MAPK.

Methods
Lysate Preparation
Lysates of HEK 293 and HeLa cells were prepared according to two techniques. The Bio-Plex cell lysis kit was used to prepare cell lysates for western blot and Bio-Plex analysis (refer to bulletin 3033). A different lysing protocol specified in the ELISA kit vendors manual was followed to prepare cell lysates for ELISA.

Assay Procedure
The procedure detailed in bulletin 3033 was followed for western blot analysis. The procedure provided in the Bio-Plex phosphoprotein detection reagent kit was followed for the Bio-Plex phosphoprotein and total t
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