Abstract
We demonstrate ultrasensitive fluorescence imaging of proteins on dot blots and western blots using a bright, compact, and orange-emitting semiconducting polymer dot (CNPPV). We achieved a detection limit at the single-picogram level in dot blots; with conventional western blot, we detected 50 pg of transferrin and trypsin inhibitor after SDS-PAGE and transfer onto a PVDF membrane. In this protocol, we describe the preparation of Pdots and its streptavidin bioconjugate and the procedures of dot blot and western blots.
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
Hawe A, Sutter M, Jiskoot W (2008) Extrinsic fluorescent dyes as tools for protein characterization. Pharm Res 25:1487–1499
Kurien BT, Scofield RH (2006) Western blotting. Methods 38:283–293
Wu C, Jin Y, Schneider T, Burnham DR, Smith PB, Chiu DT (2010) Ultrabright and bioorthogonal labeling of cellular targets using semiconducting polymer dots and click chemistry. Angew Chem Int Ed 49:9436–9440
Wu C, Schneider T, Zeigler M, Yu J, Schiro PG, Burnham DR, McNeill JD, Chiu DT (2010) Bioconjugation of ultrabright semiconducting polymer dots for specific cellular targeting. J Am Chem Soc 132:15410–15417
Ye F, Wu C, Jin Y, Chan Y-H, Zhang X, Chiu DT (2011) Ratiometric temperature sensing with semiconducting polymer dots. J Am Chem Soc 133:8146–8149
Yu J, Wu C, Zhang X, Ye F, Gallina ME, Rong Y, Wu IC, Sun W, Chan Y-H, Chiu DT (2012) Stable functionalization of small semiconducting polymer dots via covalent cross-linking and their application for specific cellular imaging. Adv Mater 24:3498–3504
Zhang X, Yu J, Rong Y, Ye F, Chiu DT, Uvdal K (2013) High-intensity near-IR fluorescence in semiconducting polymer dots achieved by cascade FRET strategy. Chem Sci 4:2143–2151
Kamaly N, Xiao Z, Valencia PM, Radovic-Moreno AF, Farokhzad OC (2012) Targeted polymeric therapeutic nanoparticles: design, development and clinical translation. Chem Soc Rev 41:2971–3010
Vonarbourg A, Passirani C, Saulnier P, Benoit J-P (2006) Parameters influencing the stealthiness of colloidal drug delivery systems. Biomaterials 27:4356–4373
Ye F, Wu C, Jin Y, Wang M, Chan Y-H, Yu J, Sun W, Hayden S, Chiu DT (2012) A compact and highly fluorescent orange-emitting polymer dot for specific subcellular imaging. Chem Commun 48:1778–1780
Ye F, Smith PB, Wu C, Chiu DT (2013) Ultrasensitive detection of proteins on western blots with semiconducting polymer dots. Macromol Rapid Commun 34:785–790
Acknowledgements
We gratefully acknowledge support of this work by the National Institutes of Health (GM085485) and the National Science Foundation (CHE-0924320). We note D.T.C. has financial interest in Lamprogen, which has licensed the described technology from the University of Washington.
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
Ye, F., Smith, P.B., Chiu, D.T. (2015). Ultrasensitive Protein Detection on Dot Blots and Western Blots with Semiconducting Polymer Dots. 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_14
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2718-0_14
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