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Quantitative NanoProteomics Approach for Protein Complex (QNanoPX) Using Gold Nanoparticle-Based DNA Probe

  • Shu-Hui Chen
  • Mei-Yin Lin
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 977)

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.

Key words

Affinity purification Pulldown Protein complex Transcription Nanoparticle Stable isotope labeling Dimethyl labeling Quantitative proteomics 

Notes

Acknowledgment

This work was supported by National Science Council in Taiwan, Republic of China, Grant NSC 99-2627-M-006-002.

References

  1. 1.
    Bonifacino JS, Dell’Angelica EC, Springer TA (2001) Immunoprecipitation. Curr Protoc Mol Biol 10:16.1–10.16.29Google Scholar
  2. 2.
    Ugelstad J, Berge A, Ellingsen T, Bjørgum J, Schmid R, Stenstad P, Aune O, Nilsen TN, Funderud S, Nustad K (1987) Biomedical applications of monodisperse magnetic polymer particles. In: El Asser MS, Fitch RM (eds) Future directions in polymer colloids. Polymer colloids in biomedical field, vol 138, NATO ASI Series E. M. Nijhoff Publ, Dodrecht, The Netherlands, pp 355–370CrossRefGoogle Scholar
  3. 3.
    Baron R, Willner B, Willner I (2007) Biomolecule-nanoparticle hybrids as functional units for nanobiotechnology. Chem Commun 28(4):323–332CrossRefGoogle Scholar
  4. 4.
    Wepf A, Glatter T, Schmidt A, Aebersold R, Gstaiger M (2009) Quantitative interaction proteomics using mass spectrometry. Nat Methods 6:203–205PubMedCrossRefGoogle Scholar
  5. 5.
    Liao LJ, Park SK, Xu T, Vanderklish P, Yates JR (2008) Quantitative proteomic analysis of primary neurons reveals diverse changes in synaptic protein content in fmr1 knockout mice. PNAS 105:15281–15286PubMedCrossRefGoogle Scholar
  6. 6.
    Cheng P-C, Chang H-K, Chen S-H (2010) Quantitative Nano-Proteomics for Protein Complexes (QNanoPX) Related to Estrogen Transcriptional Action. Mol Cell Proteomics 9:209–224PubMedCrossRefGoogle Scholar
  7. 7.
    Khan JA, Pillai B, Das TK, Singh Y, Maiti S (2007) Molecular effects of gold nanopart­icles in HeLa cells. Chembiochem 8: 1237–1240PubMedCrossRefGoogle Scholar
  8. 8.
    Murphy CJ, Gole AM, Stone JW, Sisco PN, Alkilany AM, Goldsmith DC, Baxter AC (2008) Gold nanoparticles in biology: beyond toxicity to cellular imaging. Acc Chem Res 41(12):1721–1730PubMedCrossRefGoogle Scholar
  9. 9.
    Wang A, Wu CJ, Chen SH (2006) Gold ­nanoparticle-assisted protein enrichment and electroelution for biological samples containing low protein concentrations—A prelude of gel electrophoresis. J Proteome Res 5: 1488–1492PubMedCrossRefGoogle Scholar
  10. 10.
    Jadzinsky PD, Calero G, Ackerson CJ, Bushnell DA, Kornberg RD (2007) Structure of a Thiol Monolayer–Protected Gold Nanoparticle at 1.1 Å Resolution. Science 318:430–433PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Shu-Hui Chen
    • 1
  • Mei-Yin Lin
    • 1
  1. 1.Department of ChemistryNational Cheng Kung UniversityTainanTaiwan

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