AERIUS® IMAGER
Applications for the Aerius Imaging System
ON CELL WESTERN
Application Overview
Figure 1. CB1 is internalized after exposure to a specific agonist (Win-2), but the effect is blocked by the antagonist SR11. 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).
Figure 2. Reprinted with permission from Miller, J.W.
Tracking G protein-coupled receptor trafficking using Odyssey imaging.
LI-COR Biosciences application note (2004).
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
[learn more]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
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.
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.
Further examples from the scientific literature are listed under the PubMed tab.
Techincal Resources
Protocols
See In-Cell Western™ Assay application page for related protocols.
Webinars & Video Tutorials
“Characterizing GPCR Function” -Dr. Chris Kearn, University of Washington
“The In-Cell Western™ Assay” - Dr. Amy Geschwender, LI-COR Biosciences
LI-COR documents
Kovar et al.
Purification method directly influences effectiveness of an epidermal growth factor-coupled targeting agent for noninvasive tumor detection in mice.
Anal Biochem. 361:47–54 (2007)Chen H et al.
A cell-based immunocytochemical assay for monitoring kinase signaling pathways and drug efficacy.
Anal Biochem. 338:136–142 (2005)Miller, J.W.
Tracking G protein-coupled receptor trafficking using Odyssey imaging.
LI-COR Biosciences application note (2004).Chen, H and Olive DM.
Quantitative Measurement of the Activation of Signaling Pathways Using Two-Color Infrared Fluorescent Western Blotting and Cell-Based Assays.
Poster presentation, IBC International Conference on Protein Kinases (2002)
PubMed
Odyssey® Publications: In-Cell Western™ Assays 1-2007 – 6/2009
- On-Cell Western Assays
Chan FL et al.
Functional consequence of a novel Y129C mutation in a patient with two contradictory melanocortin-2-receptor mutations.
Eur J Endocrinol. 160:705–710 (2009)
A “fluorescent cell surface assay” was among the techniques used to examine the functional consequences of an unusual mutation in the ACTH receptor. The mutant protein failed to traffic to the cell surface in this assay, a result that was confirmed by confocal microscopy.Delisle BP et al.
Small GTPase determinants for the Golgi processing and plasmalemmal expression of human Ether-a-go-go Related (hERG) K+ channels.
J Biol Chem. 284:2844-2853 (2009)
The assay was used to quantitatively measure plasmalemmal expression of hERG in live cells. The data correlated well with Western blot and whole-cell patch-clamp analyses.Chang X et al.
Role of N-linked oligosaccharides in the biosynthetic processing of the cystic fibrosis membrane conductance regulator.
J Cell Sci. 121:2814-2823 (2008)
“Cell surface ELISA” was used to examine the ability of drugs to rescue the cell surface expression of mutant CFTR protein.Chung TT et al.
The majority of ACTH receptor (MC2R) mutations found in Familial Glucocorticoid Deficiency type 1 lead to defective trafficking of the receptor to the cell surface.
J Clin Endocrinol Metab. 93(12): 4948–4954 (2008)
An immunofluorescent cell surface assay was used to study the trafficking of 23 different mutant ACTH receptors to the cell surface. Two-thirds of the mutants were found to be defective in receptor trafficking, and this conclusion was confirmed by confocal microscopy.Markovic D et al.
Intracellular mechanisms regulating Corticotropin-Releasing Hormone Receptor-2β endocytosis and interaction with Extracellularly Regulated Kinase 1/2 and p38 Mitogen-Activated Protein Kinase signaling cascades.
Mol Endocrinol. 22 (3):689–706 (2008)
The On-Cell Western assay was used to quantify the loss of the clathrin heavy chain (CRH-R2β) receptors from the cell surface after agonist stimulation.Offterdinger M and Bastiaens PI.
Prolonged EGFR signaling by ERBB2-mediated sequestration at the plasma membrane. Traffic. 9:147–155 (2008)
The recycling rate of internalized EGF was determined, using ligand labeled with an IRDye fluorophore. Wild type and mutant EGF receptors recycled internalized ligand to the extracellular space at similar rates.Gonzalez-Gronow M et al.
Plasminogen structural domains exhibit different functions when associated with cell surface GRP78 or the voltage-dependent anion channel.
J Biol Chem. 282: 32811-32820 (2007)
The On-Cell Western assay was used to quantify knockdown of glucose regulated protein 78 (GBP78) expression on the cell surface following transfection with specific siRNAs.Kovar, J et al.
Purification method directly influences effectiveness of an epidermal growth factor-coupled targeting agent for noninvasive tumor detection in mice.
Anal Biochem. 361:47-54 (2007)
A cell-based ligand binding assay was used to evaluate bioactivity of EGF labeled with an IRDye fluorophore, prior to its use as a probe for in vivo imaging. Whole-cell binding accurately predicted the ability to stimulate ERK phosphorylation and to effectively target tumors in the living animal.Egorina EM et al.
Intracellular and surface distribution of monocyte tissue factor; Application to intersubject variability.
Arterioscler Thromb Vasc Biol. 25: 1493 (2005)
“Quantitative immunofluorescent staining” was used to characterize intracellular and cell surface accumulation of monocyte tissue factor in monocytes after stimulation of activity.
- Fluorescent Ligand Binding Assays
Offterdinger M and Bastiaens PI.
Prolonged EGFR signaling by ERBB2-mediated sequestration at the plasma membrane.
Traffic. 9:147–155 (2008)
The recycling rate of internalized EGF was determined, using ligand labeled with an IRDye fluorophore. Wild type and mutant EGF receptors recycled internalized ligand to the extracellular space at similar rates.Kovar, J et al.
Purification method directly influences effectiveness of an epidermal growth factor-coupled targeting agent for noninvasive tumor detection in mice.
Anal Biochem. 361:47–54 (2007)
A cell-based ligand binding assay was used to evaluate bioactivity of EGF labeled with an IRDye fluorophore, prior to its use as a probe for in vivo imaging. Whole-cell binding accurately predicted the ability to stimulate ERK phosphorylation and to effectively target tumors in the living animal.Kovar, J et al.
A systematic approach to the development of fluorescent contrast agents for optical imaging of mouse cancer models.
Anal Biochem. 367(1):1–12 (2007)Kovar, J et al.
Hyaluronidase expression induces prostate tumor metastasis in an orthotopic mouse model.
Am J Pathol. 361:47–54 (2007)
A cell-based ligand binding assay was used to evaluate bioactivity of EGF labeled with an IRDye fluorophore, prior to its use as a probe for in vivo imaging. Whole-cell binding accurately predicted the ability to stimulate ERK phosphorylation and to effectively target tumors in the living animal.Kovar, J et al.
EGF-IRDye 800CW: in vitro and in vivo characterization as a biomarker for optical fluorescent imaging of tumor growth kinetics
Poster presentation, SMI Annual Meeting (2005)
