An On-Cell Western (OCW) Assay is a cell-based assay that enables quantitative monitoring of cell surface protein expression. It can analyze multiple samples quickly and quantitatively, and it eliminates the use of radioactivity for the assay. The OCW Assay can be used to:
With both standard In-Cell Western™ and OCW Assays, cells are first seeded in a microplate.
With In-Cell Western Assays, cell membranes are permeabilized after cells are treated and fixed, so antibodies can reach antigens inside the cell. (Not familiar with the In-Cell Western Assay? Learn more here.) However, the permeabilization process can compromise the detection of antigens present on the cell membrane.
With OCW Assays, cells are not permeabilized because the protein targets are detected on the cell surface—not intracellularly. Instead, they are stained with antibodies against extracellular protein domains, so only cell surface antigens are detected.
The cells in both assays can then be imaged either live or post-fixation.
A study by Wager Miller used the OCW Assay to study the internalization and recycling of cannabinoid receptor 1 (CB1), a G protein-coupled receptor, after treatment with receptor agonists and cycloheximide. The antibodies were targeted against specific extracellular or intracellular domains of CB1. The observed time course for receptor internalization was consistent with previous confocal microscopy studies.
A study by Royal, Tinker, & Harmer investigated the roles of two phosphatidylinositols—Phosphatidylinositol-4,5-bisphosphate (PIP2) and Phosphatidylinositol-4-phosphate (PI(4)P)—in the anterograde trafficking of KCNQ channels. They used OCW and ICW Assays to determine the surface expression (Fig. 2A) and trafficking (Fig. 2B) of VSV-KCNE1-KCNQ1 (VSV-E1-Q1), a construct whose VSV epitope was used to monitor cell-surface expression of the KCNQ1-KCNE1 (Q1/E1) channel complex.
Overall, results indicate that trafficking and expression are not affected by PIP2 and/or PI(4)P reduction at the plasma membrane or by PI(4)P reduction at the Golgi. Consequently, the Q1/E1 channel may not require PIP2 or PI(4)P for anterograde trafficking. However, PIP2 may be necessary for the activation of the Q1/E1 channel once at the plasma membrane (PM).
A study by Steel, Murray, and Liu examined the dimerization of G protein-coupled receptors (GPCRs), which help mediate human physiological responses by engaging with signaling pathways. To test a multiplex biotinylation assay, they used three chemokine receptors: CXCR4, CCR2, and CCR5. An OCW Assay was used to confirmed surface expression of the HA-tagged receptors (Fig 3). The proximity-based biotinylation assay confirmed CXCR4 homodimerization and CXCR4-CCR2 and CXCR4-CCR5 heterodimerization.
The On-Cell Western Assay is a valuable tool for quickly characterizing a broad range of cell signaling parameters in the development of targeted therapeutics. Consider using DigiWest® Protein Profiling Services for a head start in identifying potential therapeutic targets or the functional effects of a given therapeutic. Then investigate further using an On-Cell Western Assay.
See published examples of On-Cell Western AssaysView publications