title: Quantitative NanoProteomics approach for protein complex (QNanoPX) using gold nanoparticle-based DNA probe.
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.
authors: Department of Chemistry, National Cheng Kung University, Tainan, Taiwan.
authors: Shu-Hui
authors: SH
authors: Chen
authors: Mei-Yin
authors: MY
authors: Lin
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000737:chemistry; D002478:Cells, Cultured; D011232:Chemical Precipitation; D015342:DNA Probes / Q000737:chemistry*; D006046:Gold / Q000737:chemistry*; D006801:Humans; D007553:Isotope Labeling; D053768:Metal Nanoparticles / Q000737:chemistry*; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000302:isolation & purification; D011485:Protein Binding; D059748:Proteolysis; D020543:Proteome / Q000737:chemistry* / Q000302:isolation & purification; D040901:Proteomics; D020218:Response Elements; D014357:Trypsin / Q000737:chemistry
chemical_list: D015342:DNA Probes; D046912:Multiprotein Complexes; D020543:Proteome; D006046:Gold; D014357:Trypsin