An On-Cell Western (OCW) assay is a cell-based assay that enables quantitative monitoring of cell surface protein expression.
The On-Cell Western assay can be used to:
For the development of targeted therapeutics, you can get a head start by using DigiWest® Protein Profiling Services to identify therapeutic targets or functional effects before further investigation using an On-Cell Western assay.
In standard In-Cell Western™ assays, cell membranes are permeabilized so that antibodies can reach antigens inside the cell.
With On-Cell Western assays, unpermeabilized cells are stained with antibodies against extracellular protein domains, so only cell surface antigens are detected in either live or fixed cells.
Miller et al. used the assay to study internalization and recycling of CB1 (cannabinoid receptor), a G protein-coupled receptor, after treatment with receptor agonists and cycloheximide. The antibodies used were targeted against specific extracellular or intracellular domains of CB1. The observed time course of receptor internalization was consistent with previous confocal microscopy studies.
Delisle et al. adapted the assay to quantitatively measure plasmalemmal expression of hERG in live cells. The data correlated well with Western blot and whole-cell patch-clamp analyses.
Specificity is a critical parameter for signaling pathway research. This example study uses the On-Cell Western assay to demonstrate that with increased amounts of unlabeled EGF, signal from the labeled EGF decreases, allowing for confirmation of specificity and binding affinity of a ligand.
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 DigiWest® Protein Profiling Services to give you a head start in identifying potential therapeutic targets or the functional effects of a therapeutic. Then investigate further using and On-Cell Western Assay.
See some published examples of quantitative Western blotsView publications