Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

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Cadmium-113 and Lead-207 NMR Studies 213 Fig. 6. Relationship of the CaM-bound 113 Cd and 207 Pb linewidths (∆v 1/2) with the square of the magnetic field strength (B o). � and � from 207 Pb CaM peaks at δ = 983 ppm and δ = 965 ppm, respectively. � from 113 Cd CaM signal at δ = –114.5 ppm. crystal structures of some calcium-binding proteins with Cd 2+ are identical to those with Ca 2+ , lending credence to the substitution method (18). 4. Notes 1. It is not always trivial to completely remove EDTA or EGTA once they have been introduced into the protein preparation so treatment with Chelex is preferred whenever possible. Peaks from remaining EDTA or EGTA contamination can sometimes be detected in the NMR spectra. 2. For lower-affinity Ca2+ -binding sites, chemical exchange rates between free and bound Cd2+ may cause signals to be too broad for detection. On the other hand, broad resonances could result from conversion between two or more protein conformations (see Fig. 4). These two possibilities must be considered; temperature variation experiments are very useful in this respect. The chemical exchange rate for 207Pb2+ bound to calmodulin is also very temperature sensitive, with peaks disappearing above or below ambient temperature (8). 3. NMR sensitive nuclei with a large electron cloud such as 113Cd or 207Pb often relax through a mechanism known as chemical shift anisotrophy (CSA). For protein-bound nuclei, this effect becomes rather substantial and should be considered when selecting an NMR spectrometer, which will give optimal results. Figure 6 shows the consequence of CSA on 113Cd and 207Pb NMR spectra of CaM, recorded on 100 MHz, 300 MHz, 400 MHz, and 500 MHz instruments.
214 Clarke and Vogel The linewidth increases with the square of the magnetic field strength, hence, better spectra with narrower linewidths can often be obtained at somewhat lower fields. The effect is less dramatic for 113 Cd than for 207 Pb (see Fig. 6). Measurements of relaxation times are required to properly analyze these relaxation phenomena (8,9). 4. In recent years, the use of perchloric acid has been discouraged in certain laboratories. Either 113 CdSO 4 or 113 Cd(ClO 4) 2 can be successfully used as the external standard and titrant, but slight differences can be obtained in the chemical shift with various counter ions. 5. Buffers are not normally used in sample preparation for 113 Cd NMR spectroscopy since Cd 2+ ions will interact with ions such as Tris and Cl - , causing changes in linewidth and chemical shift of the resonances. It should also be noted that all NH groups on the protein will weakly interact with Cd 2+ , causing the free Cd 2+ signal to broaden. Such very weak binding sites will contribute to the exchange processes, making it difficult to obtain reasonable K d’s for a weak Cd 2+ binding site in a protein from 113 Cd NMR spectroscopy. 6. In order to properly assign the resonances, domain fragments of the protein must retain the characteriztics of the intact protein. The structure of the fragment should be similar, with the same conformational changes induced when the metal-ion binds. Often, the structure and associated metal ion induced changes of the proteolytic fragments are examined by a number of experiments to confirm their identity to each domain of the intact protein. These include 1 H NMR spectroscopy, circular dichroism experiments or Fourier transform infrared spectroscopy with titrations of each metal ion. Acknowledgments This work was supported by the Medical Research Council (MRC) of Canada and the Alberta Heritage Foundation for Medical Research (AHFMR). T. E. Clarke is supported by a Doctoral Research Award from MRC Canada and H. J. Vogel is an AHFMR Scientist. We thank Dr. T. Drakenberg and Dr. J. Aramini for collaborations and numerous insightful discussions. References 1. Vogel, H. J., Drakenberg, T., and Forsén, S. (1983) Calcium binding proteins, in NMR of Newly Accessible Nuclei (Laszlo, P., ed.), Academic, New York, pp. 157–192. 2. Forsén, S., Vogel, H. J., and Drakenberg, T. (1986) Biophysical studies of calmodulin, in Calcium and Cell Function, vol. VI (Cheung, W. Y., ed.), Academic, New York, pp. 113–157. 3. Vogel, H. J. and Forsén, S. (1987) NMR studies of calcium binding proteins. Biol. Magn. Res. 7, 245–307. 4. Drakenberg, T. (2002) Calcium-43 NMR of calcium-binding proteins, in Calcium- Binding Protein Protocols: Methods and Techniques, Vol. 2 (Vogel, H. J., ed.), Humana Press, Totowa, New Jersey, pp. 217–230.
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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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Page 181 and 182: 162 Doherty-Kirby and Lajoie metal
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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 229 and 230: 210 Clarke and Vogel Fig. 3. 113 Cd
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Page 241 and 242: 222 Drakenberg Fig. 2. Measurement
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Page 275 and 276: 256 Li et al. In order for these ex
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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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