Abstract
Affinity purification by pulldown methods using target-bound gel beads provides a powerful approach for purifying endogenous protein complexes. Such methods can be improved by using nanoparticle-based probe, coupled with immunoblot analysis or quantitative proteomics method using stable isotope labeling via liquid chromatography-mass spectrometry (LC-MS). Here, we describe sample preparation and a pulldown method using gold nanoparticle-based DNA probe for characterizing the transcriptional complex of estrogen response element (ERE). The described protocol includes the fabrication of gold nanoparticle-based probe, nuclear extract preparation, and affinity purification for the analysis by immunoblotting, as well as the subsequent trypsin digestion and stable isotope dimethyl labeling for the analysis by LC-MS.
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Acknowledgment
This work was supported by National Science Council in Taiwan, Republic of China, Grant NSC 99-2627-M-006-002.
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Chen, SH., Lin, MY. (2013). Quantitative NanoProteomics Approach for Protein Complex (QNanoPX) Using Gold Nanoparticle-Based DNA Probe. In: Bina, M. (eds) Gene Regulation. Methods in Molecular Biology, vol 977. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-284-1_14
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DOI: https://doi.org/10.1007/978-1-62703-284-1_14
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Publisher Name: Humana Press, Totowa, NJ
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Online ISBN: 978-1-62703-284-1
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