Abstract
Alternative, antibody-free techniques to western analysis of protein blots can offer reduced assay times for routine analysis of expression of recombinant proteins. We have adapted the commercially available enzyme fragment complementation technology to provide a rapid protein detection method for protein blots based on significantly reducing the number of incubation and washing steps used in traditional approaches, and eliminating the requirement for antibodies. In this chapter, we highlight the use of this assay for measuring recombinant protein expressed in mammalian cells for a range of applications, including dot blot screening of large numbers of different cell samples, assessment of protein integrity through detection of degradation bands, and characterization of posttranslational protein modifications such as glycosylation.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Kurien BT, Scofield RH (2003) Protein blotting: a review. J Immunol Methods 274:1–15
McIlhinney RA (2004) Generation and use of epitope-tagged receptors. Methods Mol Biol 259:81–98
Patton WF (2002) Detection technologies in proteome analysis. J Chromatogr B Analyt Technol Biomed Life Sci 771:3–31
Langley KE, Villarejo MR, Fowler AV, Zamenhof PJ, Zabin I (1975) Molecular basis of beta-galactosidase alpha-complementation. Proc Natl Acad Sci U S A 72:1254–1257
Eglen RM (2002) Enzyme fragment complementation: a flexible high throughput screening assay technology. Assay Drug Dev Technol 1:97–104
Zhao X, Vainshtein I, Gellibolian R, Shu Y, Dotimas H, Wang XM et al (2003) Homogeneous assays for cellular protein degradation using beta-galactosidase complementation: NF-kappaB/IkappaB pathway signaling. Assay Drug Dev Technol 1:823–833
Vainshtein I, Silveria S, Kaul P, Rouhani R, Eglen RM, Wang J (2002) A high-throughput, nonisotopic, competitive binding assay for kinases using nonselective inhibitor probes (ED-NSIP). J Biomol Screen 7:507–514
Naqvi T, Lim A, Rouhani R, Singh R, Eglen RM (2004) Beta galactosidase enzyme fragment complementation as a high-throughput screening protease technology. J Biomol Screen 9:398–408
Horecka J, Charter NW, Bosano BL, Fung P, Kobel P, Peng P et al (2006) An antibody-free method for protein detection on blots using enzyme fragment complementation. Biotechniques 40:381–383
Macian F (2005) NFAT proteins: key regulators of T-cell development and function. Nat Rev Immunol 5:472–484
Horsley V, Pavlath GK (2002) NFAT: ubiquitous regulator of cell differentiation and adaptation. J Cell Biol 156:771–774
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Charter, N.W., Horecka, J., Loh, CY., Doan, A., Wehrman, T., Olson, K.R. (2015). Rapid, Antibody-Free Detection of Recombinant Proteins on Blots Using Enzyme Fragment Complementation. In: Kurien, B., Scofield, R. (eds) Detection of Blotted Proteins. Methods in Molecular Biology, vol 1314. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2718-0_7
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2718-0_7
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2717-3
Online ISBN: 978-1-4939-2718-0
eBook Packages: Springer Protocols