Quantitative NanoProteomics Approach for Protein Complex (QNanoPX) Using Gold Nanoparticle-Based DNA Probe
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.
Key wordsAffinity purification Pulldown Protein complex Transcription Nanoparticle Stable isotope labeling Dimethyl labeling Quantitative proteomics
This work was supported by National Science Council in Taiwan, Republic of China, Grant NSC 99-2627-M-006-002.
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