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: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. 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.