Answers to Essential Questions

Answers to Essential Questions About the In‑Cell Western Assay

What Is an In‑Cell Western Assay?

An In‑Cell Western is a quantitative immunofluorescence (IF) assay performed in microwell plates (optimized for 96- or 384-well formats). Data analysis is based on whole-well fluorescence quantification. The In-Cell Western Assay offers a convenient alternative to Western blotting and is a powerful platform for meaningful in situ analyses.

What Are the Main Differences Between an In-Cell Western Assay and a Western Blot?

There are two main differences between Western blots and In‑Cell Western Assays. See TPA Dose Response | In-Cell Western | pan-ERK1 and p-ERK1/2 for an example comparison of an In‑Cell Western Assay and a Western blot assay.

Difference 1: Context in Which Proteins Are Presented As Target Antigens

In-Cell Western Assays are commonly performed on cultured cells that have been fixed to the bottom of a microwell plate and permeabilized. Antigens A substance stimulates the production of and binds to antibodies. in an In‑Cell Western often have different conformational structures than those that have been removed from the cell and processed by SDS-PAGE. Consequently, some primary antibodies that perform well for Western blotting may exhibit poor binding characteristics for fixed protein epitopes A part of the antigen to which the antibody directly binds., resulting in low or absent signal during the detection step.

Difference 2: Lack of Protein Separation

In an In-Cell Western Assay, there is no electrophoresis step to separate individual proteins by their molecular weight. Whole cells are attached to the bottom of the multiwell plate for analysis, consequently proteins are probed within the context of the entire cell. Since nonspecific antibody interactions are indistinguishable in the microplate well, antibody specificity must be confirmed prior to performing the In-Cell Western Assay. For more information about choosing the correct antibody, see "Antibody Selection".

What Advantages Does an In‑Cell Western Assay Have Over Western Blotting?

In-Cell Western Assays combine the specificity of Western blotting with the replicability and throughput of ELISA. These assays are a higher throughput way to detect and quantify multiple targets at the same time in their cellular context. Using these assays, you can rapidly collect data from many replicates under various experimental conditions. Additionally, fewer steps in the workflow mean this cell-based assay has improved consistency over traditional Western blotting.

Who Can Use an In‑Cell Western Assay?

Researchers in sectors from academia to industry can make use of In-Cell Western Assays for basic and translational research.

What Can You Use An In‑Cell Western Assay For?

These are just some of the ways an In‑Cell Western Assay can be used. Additional examples can be found in the (Undefined variable: General.licor-biosciences-biosciences) Publications Database (licorbio.com/publications).

  • Virus research including quantification of viral load (1 - 8)

  • Protein phosphorylation and signaling (9 - 11)

  • Off-target effects of drugs on signaling pathways (12)

  • Timing and kinetics of signaling events (13, 14)

  • Genotoxicity assays (15, 16)

  • Chemical library screening (17 - 19)

  • Glycoprotein analysis (20, 21)

  • Screening of monoclonal antibody clones (22)

  • Bacterial-induced epithelial signaling (23)

  • Cell proliferation and apoptosis assays (24)

Introduction References

1. Counihan, N. A., Daniel, L. M., Chojnacki, J., and Anderson, D. A. (2006). Infrared fluorescent immunofocus assay (IR-FIFA) for the quantitation of non-cytopathic and minimally cytopathic viruses. J Virol Methods, 133, 62-69. https://doi.org/10.1016/j.jviromet.2005.10.023

2. Lin, Y. C., Li, J., Irwin, C. R., Jenkins, H., DeLange, L., and Evans, D. H. (2008). Vaccinia virus DNA ligase recruits cellular topoisomerase II to sites of viral replication and assembly. J Virol, 82, 5922-5932. https://doi.org/10.1128%2FJVI.02723-07

3. Weldon, S. K., Mischnick, S. L., Urlacher, T. M., and Ambroz, K. L. (2010). Quantitation of virus using laser-based scanning of near-infrared fluorophores replaces manual plate reading in a virus titration assay. J Virol Methods, 168, 57-62. https://doi.org/10.1016/j.jviromet.2010.04.016

4. Lopez, T., Silva-Ayala, D., Lopez, S., and Arias, C. F. (2012). Methods suitable for high-throughput screening of siRNAs and other chemical compounds with the potential to inhibit rotavirus replication. J Virol Methods, 179, 242-249. https://doi.org/10.1016/j.jviromet.2011.11.010

5. Wan, Y., Zhou, Z., Yang, Y., Wang, J., and Hung, T. (2010). Application of an In-Cell Western assay for measurement of influenza A virus replication. J Virol Methods, 169, 359- 364. https://doi.org/10.1016/j.jviromet.2010.08.005

6. Fabiani, M., Limongi, D., Palamara, A. T., De Chiara, G., and Marcocci, M. E. (2017). A novel method to titrate Herpes simplex virus-1 (HSV-1) using laser-based scanning of near-infrared fluorophores conjugated antibodies. Frontiers in Microbiology, 8, 1–8. https://doi.org/10.3389/fmicb.2017.01085

7. DuShane, J. K., Wilczek, M. P., Crocker, M. A., and Maginnis, M. S. (2019). High-throughput characterization of viral and cellular protein expression patterns during JC polyomavirus infection. Frontiers in Microbiology, 10, 1–11. https://doi.org/10.3389/fmicb.2019.00783

8. Ma, H. W., Ye, W., Chen, H. S., Nie, T. J., Cheng, L. F., Zhang, L., Zhang, F. L., et al. (2017). In-cell western assays to evaluate Hantaan virus replication as a novel approach to screen antiviral molecules and detect neutralizing antibody titers. Frontiers in Cellular and Infection Microbiology, 7 https://doi.org/10.3389/fcimb.2017.00269

9. Chen, H., Kovar, J., Sissons, S., Cox, K., Matter, W., Chadwell, F., Luan, P., Vlahos, C. J., Schutz-Geschwender, A., and Olive, D. M. (2005). A cell-based immunocytochemical assay for monitoring kinase signaling pathways and drug efficacy. Analytical biochemistry, 338, 136- 142. https://doi.org/10.1016/j.ab.2004.11.015

10. Aguilar, H. N., Zielnik, B., Tracey, C. N., and Mitchell, B. F. (2010). Quantification of rapid Myosin regulatory light chain phosphorylation using high-throughput in-cell Western assays: comparison to Western immunoblots. PLoS One, 5, e9965. https://doi.org/10.1371/journal.pone.0009965

11. Wong, S. K. (2004). A 384-well cell-based phospho-ERK assay for dopamine D2 and D3 receptors. Analytical biochemistry, 333, 265-272. https://doi.org/10.1016/j.ab.2004.05.011

12. Kumar, N., Afeyan, R., Kim, H. D., and Lauffenburger, D. A. (2008). Multipathway model enables prediction of kinase inhibitor cross-talk effects on migration of Her2-overexpressing mammary epithelial cells. Mol Pharmacol, 73, 1668-1678. https://doi.org/10.1124/mol.107.043794

13. Hannoush, R. N. (2008). Kinetics of Wnt-driven beta-catenin stabilization revealed by quantitative and temporal imaging. PLoS One, 3, e3498. https://doi.org/10.1371/journal.pone.0003498

14. Chen, W. W., Schoeberl, B., Jasper, P. J., Niepel, M., Nielsen, U. B., Lauffenburger, D. A., and Sorger, P. K. (2009). Input-output behavior of ErbB signaling pathways as revealed by a mass action model trained against dynamic data. Mol Syst Biol, 5, 239. https://doi.org/10.1038/msb.2008.74

15. Jamin, E. L., Riu, A., Douki, T., Debrauwer, L., Cravedi, J. P., Zalko, D., and Audebert, M. (2013). Combined genotoxic effects of a polycyclic aromatic hydrocarbon (B(a)P) and an heterocyclic amine (PhIP) in relation to colorectal carcinogenesis. PLoS One, 8, e58591. https://doi.org/10.1371/journal.pone.0058591

16. Khoury, L., Zalko, D., and Audebert, M. (2013). Validation of high-throughput genotoxicity assay screening using gammaH2AX in-cell western assay on HepG2 cells. Environ Mol Mutagen, 54, 737-746. https://doi.org/10.1002/em.21817

17. Guo, K., Shelat, A. A., Guy, R. K., and Kastan, M. B. (2014). Development of a cell-based, high-throughput screening assay for ATM kinase inhibitors. J Biomol Screen, 19, 538-546. https://doi.org/10.1177/1087057113520325

18. Hoffman, G. R., Moerke, N. J., Hsia, M., Shamu, C. E., and Blenis, J. (2010). A high- throughput, cell-based screening method for siRNA and small molecule inhibitors of mTORC1 signaling using the In Cell Western technique. Assay Drug Dev Technol, 8, 186-199. https://doi.org/10.1089/adt.2009.0213

19. Schnaiter, S., Furst, B., Neu, J., Waczek, F., Orfi, L., Keri, G., Huber, L. A., and Wunderlich, W. (2014). Screening for MAPK modulators using an in-cell western assay. Methods Mol Biol, 1120, 121-129. https://doi.org/10.1007/978-1-62703-791-4_8

20. Urlacher, T., Xing, K., Cheung, L. et al. (2013). Glycoprotein applications using near-infrared detection. Poster presentation, Experimental Biology. https://doi.org/10.1096/fasebj.27.1_supplement.592.6

21. McInerney, M. P., Pan, Y., Short, J. L., and Nicolazzo, J. A. (2017). Development and Validation of an In-Cell Western for Quantifying P-Glycoprotein Expression in Human Brain Microvascular Endothelial (hCMEC/D3) Cells. J Pharm Sci, 106, 2614-2624. https://doi.org/10.1016/j.xphs.2016.12.017

22. Daftarian, M. P., Vosoughi, A., and Lemmon, V. (2014). Gene-based vaccination and screening methods to develop monoclonal antibodies. Methods Mol Biol. 1121, 337-346. https://doi.org/10.1007/978-1-4614-9632-8_30

23. Du, Y., Danjo, K., Robinson, P. A., and Crabtree, J. E. (2007). 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, 838-846. https://doi.org/10.1016/j.micinf.2007.03.004

24. Yunn, N-O.,Koh, A., Han, S., Lim, J.H., Park, S., Lee, J., et al. (2015). Agonistic aptamer to the insulin receptor leads to biased signaling and functional selectivity through allosteric modulation. Nucleic Acids Res, 43, 7688–7701. https://doi.org/10.1093/nar/gkv767