Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

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Steady-State Fluorescence Spectroscopy 85 b. Quenching studies with several quenching agents to obtain K SV, and then repeat the quenching experiments at several Ca 2+ ion concentrations to obtain a plot of K SV vs [Ca 2+ ]. If the calcium-binding protein interacts with another species containing a fluorophore, such as a tryptophan containing protein/ peptide, or lanthanide ion: c. Repeat the excitation and emission spectra and quenching experiments with all species varied (e.g., protein alone, peptide alone, protein + Ca 2+ , peptide + Ca 2+ , protein + peptide, protein + peptide + Ca 2+ ). If the secondary binding species is a lanthanide ion that binds in the Ca 2+ -binding region of the protein, then fluorescence of both the protein and the ion can be monitored in a competitive titration experiment. d. Red-edge emission spectral effect (REES) spectroscopy can be used to monitor different populations of Trp (14), and determining if this is a sensitive tool for following Ca 2+ -binding in titrations. e. Fluorescence energy transfer (or Förster’s resonance effect spectroscopy, FRET) can be used to monitor the distance between two specially chosen fluorophores between 10–75 Å apart (3). There is a high possibility that labeling with an extrinsic fluorophore will be necessary (5). Once a suitable system is established, quenching experiments can again be used to determine the relative solvent exposures of the two fluorophores, with and without Ca 2+ . f. Fluorescence anisotropy is another tool used to study the interaction between two biomolecules, and has successfully been applied to calcium binding systems (4). 10. A selected set of examples that encompass major fluorescence applications to calcium-binding proteins is given in Table 1. Many of these applications are dependent on specific fluorophores (e.g., unique Trp residues, or attachment sites for extrinsic fluorophores). The methods described in Table 1 also often require instrumentation and expertise beyond the scope of this chapter, but are provided for a demonstration of the usefulness of this technique. 11. Tb 3+ is a popular fluorescent Ca 2+ analogue, which can be used both intrinsically as an indicator for metal ion-binding, Ca 2+ -binding competition experiments, and also as part of a donor/acceptor pair in resonance transfer analysis. Caution should be used in assessing structure–function relationships of proteins with bound Tb 3+ however, as protein activity can be effected by this substitution. 12. Mutagenesis studies in which Tyr/Phe residues are replaced by Trp for fluorescence have been reported. It has been shown however that in the case of troponin C, such mutagenesis alters the Ca 2+ -binding properties of the protein, and results using such mutants must therefore be carefully evaluated (22,23). Acknowledgments We would like to offer our sincere gratitude to R. J. Turner for insightful discussions and suggestions. A. M. Weljie would like to thank the National Sciences and Engineering Research Council and the Alberta Heritage Fund for Medical Research (AHFMR) for support. Also, H. J. Vogel is an AHFMR Scientist.
86 Weljie and Vogel References 1. Lackowicz, J. R. (1983) Principles of Fluorescence Spectroscopy. Plenum, New York. 2. Permyakov, E. A. (1993) Luminescent Spectroscopy of Proteins. CRC, Boca Raton, Florida. 3. Selvin, P. R. (1995) Fluorescence resonance energy transfer. Methods Enzymol. 246, 300–334. 4. Jameson, D. M. and Sawyer, W. H. (1995) Fluorescence anisotropy applied to biomolecular interactions. Methods Enzymol. 246, 283–300. 5. Waggoner, A. (1995) Covalent labeling of proteins and nucleic acids with fluorophores. Methods Enzymol. 246, 362–373. 6. Chen, Y. and Barkley, M. D. (1998) Toward understanding tryptophan fluorescence in proteins. Biochemistry 37, 9976–9982. 7. Callis, P. R. (1997) 1La and 1Lb transitions of tryptophan: applications of theory and experimental observations to fluorescence of proteins. Methods Enzymol. 278, 113–150. 8. O’Neil, K. T., Wolfe, H. R., Jr., Erickson-Viitanen, S., and DeGrado, W. F. (1987) Fluorescence properties of calmodulin-binding peptides reflects alpha-helical periodicities. Science 236, 1454–1456. 9. Chabbert, M., Piemont, E., Prendergast, F. G., and Lami, H. (1995) Fluorescence of a tryptophan bearing peptide from smooth muscle myosin light chain kinase upon binding to two closely related calmodulins. Arch. Biochem. Biophys. 322, 429–436. 10. Yuan, T., Weljie, A. M., and Vogel, H. J. (1998) Tryptophan fluorescence quenching by methionine and selenomethionine residues of calmodulin: orientation of peptide and protein binding. Biochemistry 37, 3187–3195. 11. Yuan, T. and Vogel, H. J. (1998) Calcium-calmodulin-induced dimerization of the carboxyl-terminal domain from petunia glutamate decarboxylase. A novel calmodulin-peptide interaction motif. J. Biol. Chem. 273, 0328–0335. 12. Weljie, A. M. and Vogel, H. J. (1999) Tryptophan fluorescence of calmodulin binding domain peptides interacting with calmodulin containing unnatural methionine analogues. Protein Eng., in press. 13. Gill, S. C. and von Hippel, P. H. (1989) Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 182, 319. 14. Demchenko, A. P. and Ladokhin, A. S. (1988) Red-edge-excitation fluorescence spectroscopy of indole and tryptophan. Eur. Biophys. J. 15, 369–379. 15. Hogue, C., MacManus, J. P., Banville, D., and Szabo, A. G. (1992) Comparison of Terbium(III) luminescence enhancement in mutants of EF hand calcium binding proteins. J. Biol. Chem. 267, 13,340–13,347. 16. Lakowicz, J. R., Gryczynksi, I., Laczko, G., Wiczk, W., and Johnson, M. L. (1994) Distribution of distances between the tryptophan and the N-terminal residue of melittin in its complex with calmodulin, Troponin C, and phospholipids. Protein Sci. 3, 628–637.
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Methods in Molecular BiologyTM Biol
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M E T H O D S I N M O L E C U L A R
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© 2002 Humana Press Inc. 999 River
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Preface Calcium plays an important
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Contents Dedication ...............
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Contents xi 26 Enzymatic Assays to
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xiv Contents of Companion Volume 15
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xvi Contributors LESLIE D. HICKS
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20 Dean, Kelsey, and Re
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2 Yazawa
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4 Yazawa Fig. 1. Schematic represen
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6 Yazawa Fig. 2. Shop drawing for t
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8 Yazawa DuPont-NEN and the molar c
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10 Yazawa Fig. 4. Examples of the C
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12 Yazawa 5. Plot the calculated Ca
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14 Yazawa 12. Starovasnik, M. A., D
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16 Fig. 1. Molecular structures and
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18 Linse 7. 0.1 M EDTA. Dissolve 37
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20 Linse iterate the other paramete
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22 Linse tive to small alterations
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24 Linse References 1. Tsien, R. Y.
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26 Haiech and Kilhoffer to use the
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28 Haiech and Kilhoffer where γ is
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30 Haiech and Kilhoffer reporter gr
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32 Haiech and Kilhoffer tein with i
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Page 63 and 64: 44 Martin and Bayley Fig. 1. Absorp
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Page 73 and 74: 54 Martin and Bayley References 1.
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Page 79 and 80: 60 Fabian and Vogel The penetration
Page 81 and 82: 62 Fabian and Vogel perature, ionic
Page 83 and 84: 64 Fabian and Vogel Fig. 3. IR spec
Page 85 and 86: 66 Fabian and Vogel Fig. 4. Lower t
Page 87 and 88: 68 Fabian and Vogel These correlati
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136 Hicks et al. 5. Hayes, D. B. (M
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138 Trewhella and Krueger This chap
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140 Trewhella and Krueger of the sc
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142 Trewhella and Krueger Table 1 C
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144 Trewhella and Krueger beam, or
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146 Trewhella and Krueger At very l
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148 Trewhella and Krueger analytica
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150 Trewhella and Krueger collapsed
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152 Trewhella and Krueger P(r) func
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154 Trewhella and Krueger Fig. 3. S
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156 Trewhella and Krueger concentra
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158 Trewhella and Krueger by a Nati
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20 Dean, Kelsey, and Reik
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162 Doherty-Kirby and Lajoie metal
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164 Doherty-Kirby and Lajoie two Ca
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166 Doherty-Kirby and Lajoie 7. Pep
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168 Doherty-Kirby and Lajoie Fig. 1
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170 Doherty-Kirby and Lajoie Fig. 2
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172 Doherty-Kirby and Lajoie 5. Alt
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174 Doherty-Kirby and Lajoie phosph
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176 Shaw Fig. 1. Ribbon drawing of
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178 Shaw 2.3. Other 1. Chemicals fo
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180 Shaw 3.3.3. Purification 1. Con
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182 Shaw 10. Hodges, R. S., Semchuk
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184 Brokx and Vogel Table 1 An Over
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186 Brokx and Vogel Fig. 1. UV abso
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188 Brokx and Vogel Fig. 2. 20% SDS
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190 Brokx and Vogel it through an a
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192 Brokx and Vogel 8. Weljie, A. M
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196 Berliner Fig. 1. Aqueous X-band
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198 Berliner in the Bruker instrume
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200 Berliner Fig. 3. ESR spectra of
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202 Berliner Fig. 5. Low-temperatur
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204 Berliner 14. Musci, G., Reed, G
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206 Clarke and Vogel cal calcium-bi
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208 Clarke and Vogel Fig. 1. 113 Cd
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214 Clarke and Vogel The linewidth
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218 Drakenberg sands, of Hertz broa
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220 Drakenberg 1. Bloch equations m
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222 Drakenberg Fig. 2. Measurement
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224 Drakenberg Fig. 3. (A) 43 Ca NM
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226 Drakenberg Fig. 5. 43 Ca NMR sp
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228 Drakenberg NMR at sub-mM concen
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230 Drakenberg 30. Shimizu, T., Hat
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232 Weljie and Heringa Table 1 Amin
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234 Weljie and Heringa Table 2 Webs
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236 Weljie and Heringa diction meth
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238 Weljie and Heringa these databa
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240 Weljie and Heringa lineages. Th
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242 Weljie and Heringa compared, an
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244 Weljie and Heringa sequences as
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246 Weljie and Heringa much larger
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248 Weljie and Heringa ment require
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250 Weljie and Heringa 10. Kawasaki
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252 Weljie and Heringa 44. Thompson
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254 Weljie and Heringa
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256 Li et al. In order for these ex
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258 Li et al. 7. Mineral Mixture (s
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260 Li et al. 3. When the 45% D 2O/
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262 Li et al. sion system works eff
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264 Li et al. reliably produce high
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268 Mal et al. NMR data, X-PLOR (11
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270 Mal et al. Table 1 Simulated An
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272 Mal et al. 3.1.2.1. AMBIGUOUS D
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274 Mal et al. molecular dynamics a
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276 Mal et al. Assuming a rigid bod
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278 Mal et al. assign (segid A and
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280 Mal et al. References 1. Drenth
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282 Mal et al. 33. Jeener, J., Meie
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286 Werner et al. Our research has
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288 Fig. 1. (A) T 1, T 2 and the he
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290 Werner et al. tion, using gradi
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292 Werner et al. Fig. 3. (A) Rotat
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294 Werner et al. Fig. 4. (A) Line-
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296 Werner et al. Table 2 Models of
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298 Werner et al. 9. Reinhardt, D.
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300 Werner et al. 39. Press, W. H.,
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302 Boyd et al. utilize residual di
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304 Boyd et al. Fig. 1. (A) The sol
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306 Boyd et al. or significant peak
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308 Boyd et al. Fig. 3. Histogram o
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310 Boyd et al. total and NOE energ
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312 Boyd et al. 2. When studying mu
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314 Boyd et al. 4. Downing, A. K.,
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316 Boyd et al. 35. Schulte-Herbrug
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318 Yap et al. image representation
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320 Yap et al. modified EF-hand is
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322 Table 1 Angle and Distance Outp
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324 Yap et al. 2. Ikura, M. (1995)
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326 Kobayashi that S-100A1 and S-10
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328 Kobayashi 3.2. Coupling of Crom
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330 Kobayashi Fig. 2. Tricine/SDS/P
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332 Kobayashi 0.1% TFA at a flow ra
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334 Kobayashi Both recombinant S-10
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336 Kobayashi Fig. 6. Affinity chro
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340 Walsh et al. verts CaM from an
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342 Walsh et al. 11. Bovine serum a
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344 Walsh et al. Fig. 1. CaM-depend
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348 Walsh et al. 3.5. NOS Reduction
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354 Walsh et al. 3. Cho, M. J., Vag
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356 Hughes et al. The electroporati
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358 Hughes et al. 4. 1 M Na 2CO 3.
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360 Hughes et al. range, repeat ste
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362 Hughes et al. Table 1 Examples
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366 Persechini of the different CaM
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368 Persechini Fig. 1. Schematic re
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370 Persechini 2. 1 L of Terrific b
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372 Persechini Fig. 4. Titration wi
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374 Persechini Table 1 Parameters f
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376 Persechini Fig. 6. The [Ca 2+ -
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378 Persechini determined if the di
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380 Persechini relatively insensiti
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382 Persechini 18. Chafouleas, J. G
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384 Török et al. actions in the c
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386 Török et al. 3. The reaction
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388 Török et al. Fig. 2. Electros
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390 Török et al. Fig. 3. Peptides
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392 Török et al. Table 1 Peptide
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394 Török et al. Fig. 7. Nanospra
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396 Török et al. Fig. 9. Nanospra
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398 Török et al. Fig. 11. Electro
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400 Török et al. 3 µM FL-calmodu
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402 Török et al. Fig. 14. Localiz
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404 Török et al. Fig. 16. Indicat
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406 Török et al. the significantl
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408 Török et al.
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410 Index substitutes, see Fluoresc
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412 Index Stern-Volmer plot, 78, 81
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414 Index Phenothiazines, see Calmo
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