Abstract
Careful selection of well-qualified antibodies is critical for accurate data collection from reverse phase protein arrays (RPPA). The most common way to qualify antibodies for RPPA analysis is by Western blotting because the detection mechanism is based on the same immunodetection principles. Western blots of tissue or cell lysates that result in single bands and low cross-reactivity indicate appropriate antibodies for RPPA detection. Western blot conditions used to validate antibodies for RPPA experiments, including blocking and detection reagents, have significant effects on aspects of antibody performance such as cross-reactivity against other proteins in the sample. We have found that there can be a dramatic impact on antibody behavior with changes in blocking reagent and detection method, and offer an alternative method that allows detection reagents and conditions to be held constant in both antibody validation and RPPA experiments.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Grubb, R.L., Calvert, V.S., Wulkuhle, J.D., Paweletz, C.P., Linehan, W.M. et al. (2003) Signal pathway profiling of prostate cancer using reverse phase protein arrays Proteomics 3, 2142–2146.
Nishizuka, S., Carboneau, L., Young, L., Major, S., Reinhold, W.C. et al. (2003) Proteomic profiling of the NCI-60 cancer cell lines using new high-density reverse-phase lysate microarrays Proc. Natl. Acad. Sci. USA. 100, 14229–14234.
Sheehan, K.M., Calvert, V.S., Kay, E.W., Lu, Y., Fishman, D., Espina, V. et al. (2005) Use of reverse phase protein microarrays and reference standard development for molecular network analysis of metastatic ovarian carcinoma Mol. Cell. Proteomics 4,346-355.
Korf, U., Löboke, C., Sahin, Ö., Haller, F., Sültmann, H., and Poustka, A. (2009) Reverse-phase protein arrays for application-oriented cancer research Proteomics 3, 1140–1150.
Wulfkuhle, J.D., Aquino, J.A., Calvert, V.S., Fishman, D.A., Coukos, G. et al. (2003) Signal pathway profiling of ovarian cancer from human tissue specimens using reverse-phase protein microarrays Proteomics 3, 2085–2090.
Paweletz, C.P., Charboneau, L., Bichsel, V.E., Simone, N.L., Chen, T. et al. (2001) Reverse phase protein microarrays which capture disease progression show activation of pro-survival pathways at the cancer invasion front Oncogene 20,1981–1989.
Calvert, V.S., Tang, Y., Boveia, V., Wulfkuhle, J., Schutz-Geschwender, A. et al. (2004) Development of multiplexed protein profiling and detection using near infrared detection of reverse-phase protein microarrays Clin Proteomics 1, 81–89.
Towbin, H., Staehelin, T., and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Biotechnology 24, 145–149.
Aoki, H., Iwaldo, E., Eller, M., Kondo, Y. et al. (2007) Telomere 3’overhanging-specific DNA oligonucleotides induced autophagy in malignant glioma cells. FASEB J. 21, 2918–2930.
Ambroz, K.L.H., Zhang, Y., Schutz-Geschwender, A., Olive, D.M. (2008) Blocking detection chemistries affect antibody performance on reverse phase protein arrays Proteomics 8, 2379–2383.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Ambroz, K. (2011). Impact of Blocking and Detection Chemistries on Antibody Performance for Reverse Phase Protein Arrays. In: Korf, U. (eds) Protein Microarrays. Methods in Molecular Biology, vol 785. Humana Press. https://doi.org/10.1007/978-1-61779-286-1_2
Download citation
DOI: https://doi.org/10.1007/978-1-61779-286-1_2
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-285-4
Online ISBN: 978-1-61779-286-1
eBook Packages: Springer Protocols