On-Cell Western Assay

What It Is

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:

  • Detect and quantify target proteins localized to the cell surface
  • Quantify ligand binding to cell surface receptors
  • Monitor receptor internalization and recycling by detecting loss and re-appearance at the cell surface
  • Perform and detect cell surface biotinylation assays
  • Evaluate the effects of mutations, targeted therapeutics, and other treatments on protein trafficking
  • Analyze many samples quickly and quantitatively
  • Eliminate the use of radioactivity for this type of assay

How It Works

With both standard In-Cell Western (ICW) Assays and On-Cell Western Assays, cells are first seeded in a microplate.

With ICW Assays, cell membranes are permeabilized after being seeded, so antibodies can reach antigens inside the cell. However, the permeabilization process can compromise the detection of antigens present on the cell membrane.

With On-Cell Western 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.

Ways to Use It

Receptor Internalization and Recycling

Miller et al. used the On-Cell Western 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 of receptor internalization was consistent with previous confocal microscopy studies.

figure 1A
figure 1B
Effect of Win-2 agonist on CB-1 internalization in HEK293 cells measured by the OCW Assay. (A) Internalization of CB1 occurs after the addition of Win-2 agonist, as indicated by a decrease in fluorescent intensity. Treatment with SR1 antagonist recycled CB1 to the cell surface, as indicated by the restoration of the fluorescent intensity. (B) Graphical representation of the integrated intensities from Figure 1A imaged on an Odyssey® Imager.

Quantify Plasmalemmal Expression of Ion Channel

Delisle et al. adapted the On-Cell Western Assay to quantitatively measure plasmalemmal expression of hERG in live cells. The data correlated well with Western blot and whole-cell patch-clamp analyses.

Assess Binding Specificity

Specificity is a critical parameter for signaling pathway research. This example study uses an On-Cell Western Assay to demonstrate that with increased amounts of unlabeled EGF, signal from labeled EGF decreases. This consequently confirms the specificity and binding affinity of a ligand.

Targeted Therapeutics Development

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.

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