On-Cell Western Assay

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 assay

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.

How does it work?

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.

figure 1
Figure 1. CB1 is internalized after exposure to a specific agonist (Win-2), but the effect is blocked by the antagonist SR1.

figure 2
Figure 2. Panel A, fluorescent image of wells; panel B, quantification of fluorescence. Reprinted with permission from Miller, J.W. Tracking G protein-coupled receptor trafficking using Odyssey® imaging.

Uses of On-Cell Western Assays

Receptor Internalization and Recycling

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.

Quantify Plasmalemmal Expression of Ion Channel

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.

Assess Binding Specificity

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.

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 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.

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