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Fluorescent labeling and modification of proteins


This review provides an outline for fluorescent labeling of proteins. Fluorescent assays are very diverse providing the most sensitive and robust methods for observing biological processes. Here, different types of labels and methods of attachment are discussed in combination with their fluorescent properties. The advantages and disadvantages of these different methods are highlighted, allowing the careful selection for different applications, ranging from ensemble spectroscopy assays through to single-molecule measurements.

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  1. 1.

    Alivisatos AP, Gu W, Larabell C (2005) Quantum dots as cellular probes. Annu Rev Biomed Eng 7:55–76. doi:10.1146/annurev.bioeng.7.060804.100432

    Article  CAS  Google Scholar 

  2. 2.

    Altman D, Goswami D, Hasson T, Spudich JA, Mayor S (2007) Precise positioning of myosin VI on endocytic vesicles in vivo. PLoS Biol 5(8):e210. doi:10.1371/journal.pbio.0050210

    Article  Google Scholar 

  3. 3.

    Amitani I, Liu B, Dombrowski CC, Baskin RJ, Kowalczykowski SC (2010) Watching individual proteins acting on single molecules of DNA. Methods Enzymol 472:261–291. doi:10.1016/S0076-6879(10)72007-3

    Article  CAS  Google Scholar 

  4. 4.

    Antia M, Islas LD, Boness DA, Baneyx G, Vogel V (2006) Single molecule fluorescence studies of surface-adsorbed fibronectin. Biomaterials 27(5):679–690. doi:10.1016/j.biomaterials.2005.06.014

    Article  CAS  Google Scholar 

  5. 5.

    Arosio D, Ricci F, Marchetti L, Gualdani R, Albertazzi L, Beltram F (2010) Simultaneous intracellular chloride and pH measurements using a GFP-based sensor. Nat Methods 7(7):516–518. doi:10.1038/nmeth.1471

    Article  CAS  Google Scholar 

  6. 6.

    Baird GS, Zacharias DA, Tsien RY (2000) Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc Natl Acad Sci U S A 97(22):11984–11989. doi:10.1073/pnas.97.22.11984

    Article  CAS  Google Scholar 

  7. 7.

    Becker CF, Seidel R, Jahnz M, Bacia K, Niederhausen T, Alexandrov K, Schwille P, Goody RS, Engelhard M (2006) C-terminal fluorescence labeling of proteins for interaction studies on the single-molecule level. ChemBioChem 7(6):891–895. doi:10.1002/cbic.200500535

    Article  CAS  Google Scholar 

  8. 8.

    Bianco PR, Brewer LR, Corzett M, Balhorn R, Yeh Y, Kowalczykowski SC, Baskin RJ (2001) Processive translocation and DNA unwinding by individual RecBCD enzyme molecules. Nature 409(6818):374–378. doi:10.1038/35053131

    Article  CAS  Google Scholar 

  9. 9.

    Brune M, Hunter JL, Howell SA, Martin SR, Hazlett TL, Corrie JE, Webb MR (1998) Mechanism of inorganic phosphate interaction with phosphate binding protein from Escherichia coli. Biochemistry 37(29):10370–10380

    Article  CAS  Google Scholar 

  10. 10.

    Cai D, Verhey KJ, Meyhofer E (2007) Tracking single kinesin molecules in the cytoplasm of mammalian cells. Biophys J 92(12):4137–4144. doi:10.1529/biophysj.106.100206

    Article  CAS  Google Scholar 

  11. 11.

    Calleja V, Alcor D, Laguerre M, Park J, Vojnovic B, Hemmings BA, Downward J, Parker PJ, Larijani B (2007) Intramolecular and intermolecular interactions of protein kinase B define its activation in vivo. PLoS Biol 5(4):e95. doi:10.1371/journal.pbio.0050095

    Article  Google Scholar 

  12. 12.

    Campbell RE, Tour O, Palmer AE, Steinbach PA, Baird GS, Zacharias DA, Tsien RY (2002) A monomeric red fluorescent protein. Proc Natl Acad Sci U S A 99(12):7877–7882. doi:10.1073/pnas.082243699

    Article  CAS  Google Scholar 

  13. 13.

    Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC (1994) Green fluorescent protein as a marker for gene expression. Science 263(5148):802–805

    Article  CAS  Google Scholar 

  14. 14.

    Chen I, Howarth M, Lin W, Ting AY (2005) Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase. Nat Methods 2(2):99–104. doi:10.1038/nmeth735

    Article  CAS  Google Scholar 

  15. 15.

    Davidson MW, Campbell RE (2009) Engineered fluorescent proteins: innovations and applications. Nat Methods 6(10):713–717

    Article  CAS  Google Scholar 

  16. 16.

    Dickson RM, Cubitt AB, Tsien RY, Moerner WE (1997) On/off blinking and switching behaviour of single molecules of green fluorescent protein. Nature 388(6640):355–358. doi:10.1038/41048

    Article  CAS  Google Scholar 

  17. 17.

    Dong CY, French T, So PT, Buehler C, Berland KM, Gratton E (2003) Fluorescence-lifetime imaging techniques for microscopy. Methods Cell Biol 72:431–464

    Article  Google Scholar 

  18. 18.

    Elia G (2010) Protein biotinylation. Current protocols in protein science/editorial board, John E Coligan et al. (eds). doi:10.1002/0471140864.ps0306s60

  19. 19.

    Feeser EA, Ignacio CM, Krendel M, Ostap EM (2010) Myo1e binds anionic phospholipids with high affinity. Biochemistry 49(43):9353–9360. doi:10.1021/bi1012657

    Article  CAS  Google Scholar 

  20. 20.

    Fili N, Mashanov GI, Toseland CP, Batters C, Wallace MI, Yeeles JT, Dillingham MS, Webb MR, Molloy JE (2010) Visualizing helicases unwinding DNA at the single molecule level. Nucleic Acids Res 38(13):4448–4457. doi:10.1093/nar/gkq173

    Article  CAS  Google Scholar 

  21. 21.

    Galletto R, Amitani I, Baskin RJ, Kowalczykowski SC (2006) Direct observation of individual RecA filaments assembling on single DNA molecules. Nature 443(7113):875–878. doi:10.1038/nature05197

    Article  CAS  Google Scholar 

  22. 22.

    Gautier A, Juillerat A, Heinis C, Correa IR Jr, Kindermann M, Beaufils F, Johnsson K (2008) An engineered protein tag for multiprotein labeling in living cells. Chem Biol 15(2):128–136. doi:10.1016/j.chembiol.2008.01.007

    Article  CAS  Google Scholar 

  23. 23.

    Getz EB, Xiao M, Chakrabarty T, Cooke R, Selvin PR (1999) A comparison between the sulfhydryl reductants tris(2-carboxyethyl)phosphine and dithiothreitol for use in protein biochemistry. Anal Biochem 273(1):73–80. doi:10.1006/abio.1999.4203

    Article  CAS  Google Scholar 

  24. 24.

    Griffin BA, Adams SR, Tsien RY (1998) Specific covalent labeling of recombinant protein molecules inside live cells. Science 281(5374):269–272

    Article  CAS  Google Scholar 

  25. 25.

    Ha T (2001) Single-molecule fluorescence resonance energy transfer. Methods 25:78–86. doi:10.1006/meth.2001.1217

    Article  CAS  Google Scholar 

  26. 26.

    Ha T, Tinnefeld P (2012) Photophysics of fluorescent probes for single-molecule biophysics and super-resolution imaging. Annu Rev Phys Chem 63:595–617. doi:10.1146/annurev-physchem-032210-103340

    Article  CAS  Google Scholar 

  27. 27.

    Hein B, Willig KI, Wurm CA, Westphal V, Jakobs S, Hell SW (2010) Stimulated emission depletion nanoscopy of living cells using SNAP-tag fusion proteins. Biophys J 98(1):158–163. doi:10.1016/j.bpj.2009.09.053

    Article  CAS  Google Scholar 

  28. 28.

    Hohng S, Ha T (2004) Near-complete suppression of quantum dot blinking in ambient conditions. J Am Chem Soc 126(5):1324–1325. doi:10.1021/ja039686w

    Article  CAS  Google Scholar 

  29. 29.

    Howarth M, Ting AY (2008) Imaging proteins in live mammalian cells with biotin ligase and monovalent streptavidin. Nat Protoc 3(3):534–545. doi:10.1038/nprot.2008.20

    Article  CAS  Google Scholar 

  30. 30.

    Huang T, David L, Mendoza V, Yang Y, Villarreal M, De K, Sun L, Fang X, Lopez-Casillas F, Wrana JL, Hinck AP (2011) TGF-beta signalling is mediated by two autonomously functioning TbetaRI:TbetaRII pairs. EMBO J 30(7):1263–1276. doi:10.1038/emboj.2011.54

    Article  CAS  Google Scholar 

  31. 31.

    Joo C, Balci H, Ishitsuka Y, Buranachai C, Ha T (2008) Advances in single-molecule fluorescence methods for molecular biology. Annu Rev Biochem 77:51–76. doi:10.1146/annurev.biochem.77.070606.101543

    Article  CAS  Google Scholar 

  32. 32.

    Kapanidis AN, Ebright YW, Ebright RH (2001) Site-specific incorporation of fluorescent probes into protein: hexahistidine-tag-mediated fluorescent labeling with (Ni(2+):nitrilotriacetic acid (n)-fluorochrome conjugates. J Am Chem Soc 123(48):12123–12125

    Article  CAS  Google Scholar 

  33. 33.

    Kapanidis AN, Weiss S (2002) Fluorescent probes and bioconjugation chemistries for single-molecule fluorescence analysis of biomolecules. J Chem Phys 117(24):10953–10964

    Article  CAS  Google Scholar 

  34. 34.

    Lakowicz JR (2006) Principles of fluorescence spectroscopy, 3rd edn. New York, Springer

    Book  Google Scholar 

  35. 35.

    LiCata VJ, Wowor AJ (2008) Applications of fluorescence anisotropy to the study of protein–DNA interactions. Methods Cell Biol 84:243–262. doi:10.1016/S0091-679X(07)84009-X

    Article  CAS  Google Scholar 

  36. 36.

    Lin CW, Ting AY (2006) Transglutaminase-catalyzed site-specific conjugation of small-molecule probes to proteins in vitro and on the surface of living cells. J Am Chem Soc 128(14):4542–4543. doi:10.1021/ja0604111

    Article  CAS  Google Scholar 

  37. 37.

    Lippincott-Schwartz J, Patterson GH (2008) Fluorescent proteins for photoactivation experiments. Methods Cell Biol 85:45–61. doi:10.1016/S0091-679X(08)85003-0

    Article  CAS  Google Scholar 

  38. 38.

    Los GV, Encell LP, McDougall MG, Hartzell DD, Karassina N, Zimprich C, Wood MG, Learish R, Ohana RF, Urh M, Simpson D, Mendez J, Zimmerman K, Otto P, Vidugiris G, Zhu J, Darzins A, Klaubert DH, Bulleit RF, Wood KV (2008) HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem Biol 3(6):373–382. doi:10.1021/cb800025k

    Article  CAS  Google Scholar 

  39. 39.

    Manley S, Gillette JM, Patterson GH, Shroff H, Hess HF, Betzig E, Lippincott-Schwartz J (2008) High-density mapping of single-molecule trajectories with photoactivated localization microscopy. Nat Methods 5(2):155–157. doi:10.1038/nmeth.1176

    Article  CAS  Google Scholar 

  40. 40.

    Marcu L (2012) Fluorescence lifetime techniques in medical applications. Ann Biomed Eng 40(2):304–331. doi:10.1007/s10439-011-0495-y

    Article  Google Scholar 

  41. 41.

    Michalet X, Pinaud FF, Bentolila LA, Tsay JM, Doose S, Li JJ, Sundaresan G, Wu AM, Gambhir SS, Weiss S (2005) Quantum dots for live cells, in vivo imaging, and diagnostics. Science 307(5709):538–544. doi:10.1126/science.1104274

    Article  CAS  Google Scholar 

  42. 42.

    Miyawaki A, Llopis J, Heim R, McCaffery JM, Adams JA, Ikura M, Tsien RY (1997) Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388(6645):882–887

    Article  CAS  Google Scholar 

  43. 43.

    Mujumdar RB, Ernst LA, Mujumdar SR, Lewis CJ, Waggoner AS (1993) Cyanine dye labeling reagents: sulfoindocyanine succinimidyl esters. Bioconjug Chem 4(2):105–111

    Article  CAS  Google Scholar 

  44. 44.

    Nelson SR, Ali MY, Trybus KM, Warshaw DM (2009) Random walk of processive, quantum dot-labeled myosin Va molecules within the actin cortex of COS-7 cells. Biophys J 97(2):509–518. doi:10.1016/j.bpj.2009.04.052

    Article  CAS  Google Scholar 

  45. 45.

    Okada CY, Rechsteiner M (1982) Introduction of macromolecules into cultured mammalian cells by osmotic lysis of pinocytic vesicles. Cell 29(1):33–41

    Article  CAS  Google Scholar 

  46. 46.

    Phillips RA, Hunter JL, Eccleston JF, Webb MR (2003) The mechanism of Ras GTPase activation by neurofibromin. Biochemistry 42(13):3956–3965

    Article  CAS  Google Scholar 

  47. 47.

    Pierce DW, Hom-Booher N, Vale RD (1997) Imaging individual green fluorescent proteins. Nature 388(6640):338. doi:10.1038/41009

    Article  CAS  Google Scholar 

  48. 48.

    Puljung MC, Zagotta WN (2011) Labeling of specific cysteines in proteins using reversible metal protection. Biophys J 100(10):2513–2521. doi:10.1016/j.bpj.2011.03.063

    Article  CAS  Google Scholar 

  49. 49.

    Rasnik I, McKinney SA, Ha T (2006) Nonblinking and long-lasting single-molecule fluorescence imaging. Nat Methods 3(11):891–893. doi:10.1038/nmeth934

    Article  CAS  Google Scholar 

  50. 50.

    Ratner V, Kahana E, Eichler M, Haas E (2002) A general strategy for site-specific double labeling of globular proteins for kinetic FRET studies. Bioconjug Chem 13(5):1163–1170

    Article  CAS  Google Scholar 

  51. 51.

    Sakamoto T, Webb MR, Forgacs E, White HD, Sellers JR (2008) Direct observation of the mechanochemical coupling in myosin Va during processive movement. Nature 455(7209):128–132. doi:10.1038/nature07188

    Article  CAS  Google Scholar 

  52. 52.

    Shaner NC, Patterson GH, Davidson MW (2007) Advances in fluorescent protein technology. J Cell Sci 120(Pt 24):4247–4260. doi:10.1242/jcs.005801

    Article  CAS  Google Scholar 

  53. 53.

    Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nat Methods 2(12):905–909. doi:10.1038/nmeth819

    Article  CAS  Google Scholar 

  54. 54.

    Shanker N, Bane SL (2008) Basic aspects of absorption and fluorescence spectroscopy and resonance energy transfer methods. Methods Cell Biol 84:213–242

    Article  CAS  Google Scholar 

  55. 55.

    Shimomura O, Johnson FH, Saiga Y (1962) Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol 59:223–239

    Article  CAS  Google Scholar 

  56. 56.

    Shu X, Lev-Ram V, Deerinck TJ, Qi Y, Ramko EB, Davidson MW, Jin Y, Ellisman MH, Tsien RY (2011) A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms. PLoS Biol 9(4):e1001041. doi:10.1371/journal.pbio.1001041

    Article  CAS  Google Scholar 

  57. 57.

    Shu X, Shaner NC, Yarbrough CA, Tsien RY, Remington SJ (2006) Novel chromophores and buried charges control color in mFruits. Biochemistry 45(32):9639–9647. doi:10.1021/bi060773l

    Article  CAS  Google Scholar 

  58. 58.

    Sun X, Zhang A, Baker B, Sun L, Howard A, Buswell J, Maurel D, Masharina A, Johnsson K, Noren CJ, Xu MQ, Correa IR Jr (2011) Development of SNAP-tag fluorogenic probes for wash-free fluorescence imaging. ChemBioChem 12(14):2217–2226. doi:10.1002/cbic.201100173

    Article  CAS  Google Scholar 

  59. 59.

    Theissen B, Karow AR, Kohler J, Gubaev A, Klostermeier D (2008) Cooperative binding of ATP and RNA induces a closed conformation in a DEAD box RNA helicase. Proc Natl Acad Sci U S A 105(2):548–553. doi:10.1073/pnas.0705488105

    Article  CAS  Google Scholar 

  60. 60.

    Toseland CP, Martinez-Senac MM, Slatter AF, Webb MR (2009) The ATPase cycle of PcrA helicase and its coupling to translocation on DNA. J Mol Biol 392(4):1020–1032

    Article  CAS  Google Scholar 

  61. 61.

    Toseland CP, Powell B, Webb MR (2012) ATPase cycle and DNA unwinding kinetics of RecG helicase. PLoS One 7(6):e38270. doi:10.1371/journal.pone.0038270

    Article  CAS  Google Scholar 

  62. 62.

    Tsien RY (1998) The green fluorescent protein. Annu Rev Biochem 67:509–544. doi:10.1146/annurev.biochem.67.1.509

    Article  CAS  Google Scholar 

  63. 63.

    Vogelsang J, Kasper R, Steinhauer C, Person B, Heilemann M, Sauer M, Tinnefeld P (2008) A reducing and oxidizing system minimizes photobleaching and blinking of fluorescent dyes. Angew Chem Int Ed Engl 47(29):5465–5469. doi:10.1002/anie.200801518

    Article  CAS  Google Scholar 

  64. 64.

    Waggoner A (1995) Covalent labeling of proteins and nucleic acids with fluorophores. Methods Enzymol 246:362–373

    Article  CAS  Google Scholar 

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Thanks Dr. Natalia Fili for helpful comments on the manuscript and EMBO for funding.

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Correspondence to Christopher P. Toseland.

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Toseland, C.P. Fluorescent labeling and modification of proteins. J Chem Biol 6, 85–95 (2013).

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  • Fluorescence
  • Single molecule
  • Fluorophores
  • Fluorescent proteins
  • Quantum dots