Application Overview

What is the In-Cell Western™ (ICW)?

A quantitative immunofluorescent assay for simultaneous analysis of two different proteins in fixed cultured cells.

Advantages

In-Cell Western™ assays provide many unique advantages:

• Sensitive, quantitative analysis of cellular signaling pathways

  • Reduced autofluorescent interference from cells, microplates,
    and chemical compounds

• “Snapshot” of signal transduction or protein expression of cell population in each well

• Enhanced throughput

  • Eliminate cell lysis, gel loading, electrophoresis, and membrane transfer

  • Streamline the experimental procedure and data analysis to easily process
    many samples at once

• Increased precision

  • Use ratiometric analysis to normalize for variations in cell number

  • Quantify small changes in protein levels reliably

• Easily screen cell treatments or drug candidates for their effects on target proteins

• Obtain more relevant results than enzyme assays with purified proteins

Workflow

Support Materials

Need help identifying a primary antibody?

*For resources related to individual steps of the In-Cell Western assay, see the Workflow tab.

Brochures, Protocols and Documents:

• In-Cell Western Assays Brochure

• In-Cell Western Kits I & II Protocol

• Printable Microplate Set-Up Guide

• "Quantitative Methods for analysis of protein phosphorylation in drug development"
  Olive, D.M. Expert Rev. Proteomics 1:327–341 (2004)

• "Quantitative Measurement of the Activation of Signaling Pathways Using Two-Color Infrared Fluorescent Western Blotting and Cell-Based Assays"
  Chen, H. and D.M. Olive, poster presentation, IBC International Conference on Protein Kinases (2002)

• "A cell-based immunocytochemical assay for monitoring kinase signaling pathways and drug efficacy"
  H. Chen, et al. Anal. Biochem. 338:136-142 (2005)

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Webinars & Video Tutorials:

• “The In-Cell Western™ Assay”
  Dr Amy Schutz-Geschwender, LI-COR

• “Characterizing GPCR Function”
  Dr Chris Kearn, University of Washington

• “In-Cell Western^™ (ICW) Assay; Power User Guide”
  Ashlee Muller, LI-COR

• “In-Cell Western^™ Assay: From Cell Proliferation to Protein Trafficking"
  Dr Harry Osterman, LI-COR

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PubMed

Publishing with LI-COR data?

Odyssey® Publications: In-Cell Western™ Assays Summer 2011

Odyssey Publications: RNAi interference 1/2007-6/2009

• Analysis of the kinetics of cell signaling and phosphorylation

  • Kumar et al.
    Multipathway model enables prediction of kinase inhibitor cross-talk effects on migration of Her2-overexpressing mammary epithelial cells.
    Mol Pharmacol 73:1668–1678 (2008)
    The authors evaluated a method for predictive modeling of “off-target” and “pathway crosstalk” effects of small molecule kinase inhibitors. They used the ICW assay to perform quantitative, multipathway measurements of signaling events in a higher-throughput manner. With this method, they were able to predictively understand how kinase inhibitor treatment influences cell migration behavior.

  • Hannoush, RN.
    Kinetics of Wnt-driven β-catenin stabilization revealed by quantitative and temporal imaging.
    PLoS ONE 3(10):e3498 (2008)
    The author used the ICW assay as a high-throughput, quantitative method for measuring Wnt-induced changes in β-catenin protein levels. The assay revealed an unprecedented level of detail about the kinetics of β-catenin accumulation. The author suggests that the assay would be appropriate for secondary screening to quickly determine whether inhibitors of Wnt/β-catenin signaling act upstream or downstream of β-catenin.

  • Chen, WW et al.,
    Input–output behavior of ErbB signaling pathways as revealed by a mass action model trained against dynamic data.
    Mol Syst Biol 5:239 (2009)

  • Du, Y et al.
    In-Cell Western analysis of Helicobacter pylori-induced phosphorylation of extracellular-signal related kinase via the transactivation of the epidermal growth factor receptor
    Microbes Infect 9(7):838-46 (2007)
    Du et al developed an ‘In-Cell Western’ (ICW) assay for quantitative examination of H. pylori-induced epithelial signalling to determine the role of the EGF receptor (EGFR) in H. pylori-induced phosphorylation of ERK in epithelial cells. Helicobacter pylori activates extracellular-signal related (ERK) kinases in gastric epithelial cells via transactivation of EGFR. H. pylori activation of EGFR may be relevant to epithelial hyperproliferation and gastric carcinogenesis. H. pylori strains were co-incubated with A431 and AGS cells. pERK and total ERK were quantified in situ using ICW analysis. The EGFR inhibitor EKB-569 dose-dependently reduced H. pylori-induced ERK phosphorylation in A431 and AGS cells. The data suggested that H. pylori induces pERK in epithelial cells partly via the EGFR pathway. Additional signalling mechanisms are likely to be involved in H. pylori-induced ERK phosphorylation. ICW analysis is a rapid quantitative method for evaluating the effects of inhibitors on H. pylori-induced cell signalling pathways of relevance to gastric carcinogenesis.

  • Wong, S.
    A 384-well cell-based phosho-ERK assay for dopamine D2 and D3 receptors
    Anal Biochem. 333(2): 265-72 (2004)

• Quantification of “off-target” drug effects in signaling pathway analysis

  • Kumar, N et al.
    Multipathway model enables prediction of kinase inhibitor cross-talk effects on migration of Her2-overexpressing mammary epithelial cells.
    Mol Pharmacol. 73(6):1668–78 (2008).
    The authors evaluated a method for predictive modeling of “off-target” and “pathway crosstalk” effects of small molecule kinase inhibitors. They used the ICW assay to perform quantitative, multipathway measurements of signaling events in a higher-throughput manner. With this method, they were able to predictively understand how kinase inhibitor treatment influences cell migration behavior.

• Cell-based determinations of IC50 concentrations and efficacy testing of drug compounds

  • Maira SM et al.
    Identification and characterization of NVP-BEZ235, a new orally available dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor with potent in vivo antitumor activity.
    Mol Cancer Ther. 7(7):1851-63 (2008)

  • Yeh et al.
    Biological characterization of ARRY-142886 (AZD6244), a potent, highly selective mitogen-activated protein kinase kinase 1/2 inhibitor.
    Clin Cancer Res 13(5):1576-83 (2007)

  • Chen et al.
    A cell-based immunocytochemical assay for monitoring kinase signaling pathways and drug efficacy.
    Analytical Biochemistry 338:136–142 (2005)

• Cell surface protein analysis and quantification

  • see On-Cell Western
    

• Receptor trafficking and recycling assays

  • see On-Cell Western
    

• Monitoring of GPCR activation

  • Selkirk, JV et al.
    A novel cell-based assay for G-protein-coupled receptor-mediated cyclic adenosine monophosphate response element binding protein phosphorylation.
    J Biomol Screen. 11(4): 351-8 (2006)
    The authors validated the use of ICWs as a quantitative readout of GPCR function. Specifically, they used CREB phosphorylation as a downstream readout of GPCR activity in whole cells after treatment with receptor agonists. They validated this as a reliable high-throughput readout by comparing the results to traditional radioligand binding assays and cAMP competitive immunoassays.

  • Wacker, JL et al.
    Disease-causing mutation in GPR54 reveals the importance of the second intracellular loop for class A G-protein-coupled receptor function.
    J Biol Chem. 283(45):31068-78 (2008)

• Cell proliferation and cell number assays

  • Widberg, CH et al.
    Fibroblast growth factor receptor 1 is a key regulator of early adipogenic events in human preadipocytes.
    Am J Physiol Endocrinol Metab. 296(1):E121-31 (2009).

  • Kim, WJ et al. Blood.
    Mutations in the neutral sphingomyelinase gene SMPD3 implicate the ceramide pathway in human leukemias.
    111(9):4716-22 (2008).

  • Godin-Heymann, N et al.
    The T790M “gatekeeper” mutation in EGFR mediates resistance to low concentrations of an irreversible EGFR inhibitor.
    Mol Cancer Ther. 7(4):874-9 (2008)

  • Quinlan, MP et al.
    Activated Kras, but Not Hras or Nras, May Initiate Tumors of Endodermal Origin via Stem Cell Expansion.
    Mol Cell Biol. 28(8):2659-74 (2008)

• Surface protein expression on living cells

  • 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(5):2844-53 (2009)
    Delisle et al developed a ‘live-cell’ Western assay that quantitatively measures plasmalemmal expression of hERG by imaging living cells on tissue culture plates. This assay employs an antibody against an extracellular epitope in the hERG S1-S2 linker which is then detected by a IRDye 700 secondary antibody on Odyssey. These live-cell Western data recapitulates the findings from the Western blot, whole-cell patch clamp, and immunocytochemical analyses.

• Determination of viral titer and infectivity

  • Lin, YC et al.
    Vaccinia virus DNA ligase recruits cellular topoisomerase II to sites of viral replication and assembly.
    J Virol. 82: 5922-32 (2008)
    The In-Cell Western assay was used to image viral plaque size and number in a study of the role of cellular topoisomerases in vaccinia virus replication and assembly.

  • Sklan et al,
    A Rab-GAP TBC Domain Protein Binds Hepatitis C Virus NS5A and Mediates Viral Replication.
    J Virol. 81: 11096–11105 (2007)

  • Counihan, NA et al.
    Infrared fluorescent immunofocus assay (IR-FIFA) for the quantitation of non-cytopathic and minimally cytopathic viruses.
    J Virol. 133:62-69 (2006)

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