Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

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Calcium-43 NMR 227 Fig. 6. 43 Ca NMR linewidth as a function of temperature in the absence (open circles) and presence (filled circles) of 20 mM Mg 2+ , pH 7.2 in 100 mM KCl. The upper-dashed curve shows the linewidth calculated using activation parameters from ref. 20. The lower dashed curve has been calculated with the same parameters with the population of free Ca 2+ adjusted to obtain the proper linewidth at 25°C. The best agreement with experimental linewidth, as shown by the solid curve was obtained using a quadrupolar coupling χ = 1.22 MHz and an off-rate at 25°C of 6600 s –1 (compared to 500 s –1 for the upper curve). faster than the relaxation rate of the bound ions (19,35–37). It is thus possible to determine Ca 2+ off-rates from approx 10 s –1 to approx 10 5 s –1 . This means that 43 Ca NMR can determine rates 100 times faster than those that can be obtained from stopped-flow studies. We have, in Lund, used the scheme outlined above on several occasions to study the Ca 2+ exchange. Most recently, we have applied it in a study of the Ca–Mg competition for the calcium-binding sites in the N-terminal domain of calmodulin (25). As shown in Fig. 6, it was possible to show that the exchange rate of Ca 2+ from the Ca 1 Mg 1-form of TR 1C from calmodulin is much faster than from the Ca 2-form. This is completely in agreement with the strong cooperativity in calcium binding and the lack of cooperativity between calcium and magnesium. I hope that we have been able to show that 43 Ca NMR can be quite useful on many occasions when studying calcium-binding proteins. It is disappointing that so few groups have been considering the use of it to date. With the high sensitivity of modern NMR spectrometers, it should be possible to run 43 Ca
228 Drakenberg NMR at sub-mM concentration without having probes specifically designed for this particular purpose. Even though the cost for enriched 43 Ca may appear high, the total cost for a sample will almost always be dominated by the cost involved in preparation of the protein. Hopefully, in the future, more groups will consider the use of 43 Ca NMR in their studies of calcium-binding proteins. References 1. Robertson, P., Hiskey, R. G., and Koehler, K. L. (1978) Calcium and magnesium binding to γ-carboxyglutamic acid-containing peptides via metal ion nuclear magnetic resonance. J. Biol. Chem. 253, 5880–5883. 2. Andersson, T., Drakenberg, T., Forsén, S., Thulin, E., and Svärd, M. (1982) Direct observation of the 43Ca ions bound to proteins. J. Am. Chem. Soc. 104, 576–580. 3. Shimizu, T. and Hatano, M. (1985) Magnetic resonance studies of trifluoperazinecalmodulin solutions: 43Ca, 25Mg, 67Zn, and 39K nuclear magnetic resonance. Inorg. Chem. 24, 2003–2009. 4. Urry, D. W., Trapane, T. L., and Venkatachalam, C. M. (1982) Calcium binding to a calcifiable matrix: 43Ca NMR binding studies on the polypentapeptide of elastin. Calcif. Tissue Int. 34, S41–S46. 5. Bouhoutsos-Brown, E., Pletcher, C. H., Nelsestuen, G. L., and Bryant, R. G. (1984) Prothrombin fragment 1-membrane interactions: a calcium-43 NMR study. J. Inorg. Biochem. 21, 337–343. 6. Aramini, J. H., Drakenberg, T., Hiraoki, T., Nitta, K., and Vogel, H. (1992) Calcium-43 NMR studies of calcium-binding lysozymes and α-lactalbumins. Biochemistry 31, 6761–6768. 7. Belton, P. S., Cox, I. Y., and Harris, R. K. (1985) Experimental sulphur-33 nuclear magnetic resonance spectroscopy. J. Chem. Soc. Faraday, Trans 2 81, 63–75. 8. Ernst, R. R., Bodenhusen, G., and Wokaun, A. (1989) Principles of Nuclear Magnetic Resonance in One and Two Dimensions. Oxford Science, Oxford, United Kingdom. 9. Bull, T. E., Forsén, S., and Turner, D. L. (1978) Nuclear magnetic relaxation of spin 5/2 and 7/2 nuclei including effect of chemical exchange. J. Chem. Phys. 70, 3106–3111. 10. Halle, B. and Wennerström, H. (1981) Nearly exponential quadrupole relaxation. A perturbation treatment. J. Magn. Reson. 44, 89–100. 11. Bull, T. E. (1972) Nuclear magnetic relaxation of spin-3/2 nuclei involved in chemical exchange. J. Magn. Reson. 8, 344–353. 12. Drakenberg, T., Johansson, C., and Forsén, S. (1997) Metal NMR for the study of metalloproteins, in Protein NMR Techniques (Reid, D. G., ed.), Humana, Totowa, New Jersey. 13. Sandström, J. (1982) Dynamic NMR Spectroscopy, Academic, London. 14. McConnell, H. M. (1958) Reaction rates by nuclear magnetic resonance. J. Magn. Reson. 28, 430–431.
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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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34 Haiech and Kilhoffer Calmodulin
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36 Haiech and Kilhoffer Fig. 3. Mec
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38 Haiech and Kilhoffer We may sugg
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40 Haiech and Kilhoffer 29. Stewart
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42 Haiech and Kilhoffer 62. Becking
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44 Martin and Bayley Fig. 1. Absorp
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46 Martin and Bayley evaporation. C
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48 Martin and Bayley 4. Set the tem
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50 Martin and Bayley Fig. 2. Near-U
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52 Martin and Bayley discussed by M
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54 Martin and Bayley References 1.
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20 Dean, Kelsey, and Reik
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58 Fabian and Vogel are equipped wi
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60 Fabian and Vogel The penetration
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62 Fabian and Vogel perature, ionic
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64 Fabian and Vogel Fig. 3. IR spec
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66 Fabian and Vogel Fig. 4. Lower t
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68 Fabian and Vogel These correlati
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70 Fabian and Vogel Fig. 5. Amide I
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72 Fabian and Vogel 4. ATR-FTIR spe
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74 Fabian and Vogel 25. Georg, H.,
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76 Weljie and Vogel Fig. 1. Simplif
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78 Weljie and Vogel Fig. 3. Steady-
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80 Weljie and Vogel VIS spectrophot
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82 Weljie and Vogel 4. A series of
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84 Table 1 Advanced Fluorescence Me
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86 Weljie and Vogel References 1. L
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90 Johnson and Tikunova is removed
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92 Johnson and Tikunova function of
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94 Johnson and Tikunova Fig. 2. Rat
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96 Johnson and Tikunova Fig. 3. Rat
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98 Johnson and Tikunova Fig. 4. Ca
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100 Johnson and Tikunova cence inte
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102 Johnson and Tikunova 6. Johnson
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104 Julenius Fig. 1. Under conditio
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106 Julenius 3. Mix 50 µL of NHS s
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108 Julenius Fig. 3. A typical sens
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110 Julenius (2), is used in the Ia
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114 Lopez and Makhatadze Fig. 1. Th
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116 Lopez and Makhatadze 6. Buffers
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118 Lopez and Makhatadze The excess
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122 Lopez and Makhatadze Fig. 1. Is
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124 Lopez and Makhatadze 2.5 mL are
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126 Lopez and Makhatadze pendence o
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128 Hicks et al. equipped with a pu
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130 Hicks et al. Fig. 2. Debye plot
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132 Hicks et al. and 1.0 mg/mL for
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134 Hicks et al. Fig. 3. Sedimentat
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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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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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Page 203 and 204: 184 Brokx and Vogel Table 1 An Over
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Page 215 and 216: 196 Berliner Fig. 1. Aqueous X-band
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Page 225 and 226: 206 Clarke and Vogel cal calcium-bi
Page 227 and 228: 208 Clarke and Vogel Fig. 1. 113 Cd
Page 233 and 234: 214 Clarke and Vogel The linewidth
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Page 239 and 240: 220 Drakenberg 1. Bloch equations m
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Page 269 and 270: 250 Weljie and Heringa 10. Kawasaki
Page 271 and 272: 252 Weljie and Heringa 44. Thompson
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Page 275 and 276: 256 Li et al. In order for these ex
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Page 279 and 280: 260 Li et al. 3. When the 45% D 2O/
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Page 289 and 290: 270 Mal et al. Table 1 Simulated An
Page 291 and 292: 272 Mal et al. 3.1.2.1. AMBIGUOUS D
Page 293 and 294: 274 Mal et al. molecular dynamics a
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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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