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Eastern Staining: A Simple Recombinant Protein Detection Technology Using a Small Peptide Tag and Its Counter Partner Which is a Fluorescent Compound

  • Lee, Jae-Jung (Laboratory of Bioimaging Probe Development, Singapore Bioimaging Consortium, Agency for Science, Technology and Research) ;
  • Kim, Jun-Young (Department of Chemistry, MedChem Program of Life Sciences Institute, National University of Singapore) ;
  • Zhai, Duanting (Department of Chemistry, MedChem Program of Life Sciences Institute, National University of Singapore) ;
  • Yun, Seong-Wook (Laboratory of Bioimaging Probe Development, Singapore Bioimaging Consortium, Agency for Science, Technology and Research) ;
  • Chang, Young-Tae (Laboratory of Bioimaging Probe Development, Singapore Bioimaging Consortium, Agency for Science, Technology and Research)
  • Received : 2012.06.01
  • Accepted : 2012.06.15
  • Published : 2012.06.30

Abstract

Small peptide tags such as c-myc, HA, or FLAG tag have facilitated efficient Western-blotting of proteins of interest especially when specific antibodies for the proteins are not available. However, the conventional Western-blotting requires the multi-steps process taking at least several hours up to two days. With examples of various applications, here we show a convenient and time-saving method for protein detection which employs a fluorescent chemical BDED and its binding peptide RC-tag. And we propose "Estern staining", as a standard term for protein detection method using fluorescent chemicals and their binding small peptide tags. Eastern staining may substitutes for the time-consuming "immuno-staining" in many versatile applications.

Keywords

References

  1. Brizzard, B. (2008). Epitope tagging. BioTechniques 44, 693-695. https://doi.org/10.2144/000112841
  2. Terpe, K. (2003). Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol 60, 523-533. https://doi.org/10.1007/s00253-002-1158-6
  3. Evan, G.I., Lewis, G.K., Ramsay, G., and Bishop, J.M. (1985). Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol 5, 3610-3616. https://doi.org/10.1128/MCB.5.12.3610
  4. Einhauer, A., and Jungbauer, A. (2001). The FLAG peptide, a versatile fusion tag for the purification of recombinant proteins. J Biochem Biophys Meth 49, 455-465. https://doi.org/10.1016/S0165-022X(01)00213-5
  5. Prescher, J.A., and Bertozzi, C.R. (2005). Chemistry in living systems. Nat Chem Biol 1, 13-21. https://doi.org/10.1038/nchembio0605-13
  6. Griffin, B.A., Adams, S.R., and Tsien, R.Y. (1998). Specific covalent labeling of recombinant protein molecules inside live cells. Science 281, 269-272. https://doi.org/10.1126/science.281.5374.269
  7. Girouard, S., Houle, M.H., Grandbois, A., Keillor, J.W., and Michnick, S.W. (2005). Synthesis and characterization of dimaleimide fluorogens designed for specific labeling of proteins. J Am Chem Soc 127, 559-566. https://doi.org/10.1021/ja045742x
  8. Lee, J.J., Lee, S.C., Zhai, D., Ahn, Y.H., Yeo, H.Y., Tan, Y.L., and Chang, Y.T. (2011). Bodipy-diacrylate imaging probes for targeted proteins inside live cells. Chem Commun (Camb) 47, 4508-4510. https://doi.org/10.1039/c1cc10362h
  9. Einarson, M.B. (2001). In Molecular Cloning: A Laboratory Manual, 3rd Edition (Cold Spring Harbor Kaboratory Press).
  10. Hengen, P. (1995). Purification of His-Tag fusion proteins from Escherichia coli. Trends Biochem Sci 20, 285-286. https://doi.org/10.1016/S0968-0004(00)89045-3
  11. Donovan, R.S., Robinson, C.W., and Glick, B.R. (1996). Review: optimizing inducer and culture conditions for expression of foreign proteins under the control of the lac promoter. J Ind Microbiol 16, 145-154. https://doi.org/10.1007/BF01569997
  12. Krishnan, B., Szymanska, A., and Gierasch, L.M. (2007). Site-specific fluorescent labeling of poly-histidine sequences using a metal-chelating cysteine. Chem Biol Drug Des 69, 31-40. https://doi.org/10.1111/j.1747-0285.2007.00463.x
  13. Zhao, C., Hellman, L.M., Zhan, X., Bowman, W.S., Whiteheart, S.W., and Fried, M.G. (2010). Hexahistidine-tag-specific optical probes for analyses of proteins and their interactions. Anal Biochem 399, 237-245. https://doi.org/10.1016/j.ab.2009.12.028
  14. Los, G.V., Encell, L.P., McDougall, M.G., Hartzell, D.D., Karassina, N., Zimprich, C., Wood, M.G., Learish, R., Ohana, R.F., Urh, M., et al. (2008). HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem Biol 3, 373-382. https://doi.org/10.1021/cb800025k
  15. Keppler, A., Gendreizig, S., Gronemeyer, T., Pick, H., Vogel, H., and Johnsson, K. (2003). A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat Biotechnol 21, 86-89. https://doi.org/10.1038/nbt765
  16. Chen, I., Howarth, M., Lin, W., and Ting, A.Y. (2005). Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase. Nat Methods 2, 99-104. https://doi.org/10.1038/nmeth735
  17. Fernandez-Suarez, M., Baruah, H., Martinez-Hernandez, L., Xie, K.T., Baskin, J.M., Bertozzi, C.R., and Ting, A.Y. (2007). Redirecting lipoic acid ligase for cell surface protein labeling with small-molecule probes. Nat Biotechnol 25, 1483-1487. https://doi.org/10.1038/nbt1355