Receptor-based Binding and Competition Assays with Infrared Fluorescence

What is an On-Cell Western? It’s a cell-based assay that enables quantitative monitoring of cell surface protein expression. The On-Cell Western assay 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

On-Cell Western Assay Example with Quantification

Figure 1. Cannabinoid receptor 1 (CB1) is internalized after exposure to a specific agonist (Win-2), but the effect is blocked by the antagonist SR1. A) Intensity levels were greatly reduced in wells treated with 1μM of the CB1-specific agonist, Win-2. Cells treated with Win-2 and the specific CB1 antagonist, SR1 displayed no reduction of signal with the treatment. B) Graph displaying results of three independent experiments done in quadruplicate.

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

Visit On-Cell Westerns for more information. We have a sample protocol of On-Cell Western Assay for Targeted Near-infrared-labeled optical imaging agent development. For other scientific publications in which On-Cell Westerns were used, visit our On-Cell Westerns Publications page.

Updated February 18, 2015.

Monitor Cell Surface Protein Expression with Infrared Fluorescence Detection

Are you studying receptor binding or cell surface protein expression? The On-Cell Western Assay is a great way to monitor cell surface proteins – quantitatively! – with near-infrared fluorescent detection. Imaging can be performed on the Odyssey® CLx, Odyssey Classic, or the Odyssey Sa Infrared Imaging System.

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 analyses use detection at the well surface with no liquid present. This results in minimal well-to-well signal spread, allowing the use of both clear and black-sided plates with clear bottoms. Do not use plates with white wells, since the autofluorescence from the white surface will create significant noise.

Here is some data from a white paper by J. W. Miller:

Schematic of Cannabinoid Receptor in a cell membrane.

Figure 1. Antibodies used were targeted against specific extracellular or intracellular domains of the CB1 (cannabinoid 1) receptor. Reprinted with permission from Miller, J.W. Tracking G protein-coupled receptor trafficking using Odyssey imaging.


On-Cell Western Assay showing effects of cannabinoid-specific agonist on CB1 receptors.

Figure 2. Intensity levels were greatly reduced in wells treated with 1 μM of the CB1-specific agonist, Win-2. Cells treated with Win-2 and the specific CB1 antagonist, SR1 displayed no reduction of signal with the treatment. B) Graph displaying results of three independent experiments done in quadruplicate. Reprinted with permission from Miller, J.W. Tracking G protein-coupled receptor trafficking using Odyssey imaging.