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.