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On-Cell Western Assay

What is the On-Cell Western?

A cell-based assay that enables quantitative monitoring of cell surface protein expression. The On-Cell Western offers the ability 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, drugs, and other treatments on protein trafficking
  • Analyze many samples quickly and quantitatively
  • Avoid use of radioactivity
Cell surface receptors detected with fluorescent dye-based On-Cell Western Assays on the Odyssey Imager

Figure 1. CB1 is internalized after exposure to a specific agonist (Win-2), but the effect is blocked by the antagonist SR1. 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. LI-COR Biosciences application note (2004).

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.
on cell western

Figure 2. Reprinted with permission from Miller, J.W. Tracking G protein-coupled receptor trafficking using Odyssey imaging. LI-COR Biosciences application note (2004).

How is it used?

  • 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.
  • See Publications for further examples from scientific literature.
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