Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

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<strong>Calcium</strong>-43 NMR 225 Fig. 4. Dependence of the 43 Ca NMR linewidth on pH at 23°C; (A) 0.98 mM phospholipae A 2 and 1.1 mM 43 Ca 2+ ; (B) 1.1 mM phospholipase A 2 and 11.5 mM 43 Ca 2+ . The dashed curves are calculated using Eq. 6 and the solid curves are calculated taking two binding sites into account. Reproduced with permission from ref. 17. where [E] = [Etot]/(1 + K[Ca2+ ] + 10pKa-pH ) and [Ca2+ ] = [Catot]/(1 + K[E]) Equation 6 can also be extended to take more than one binding site or pKa into account (Fig. 4) (17). 43Ca NMR has also been used to study the effect of drug binding on the calcium-binding properties of proteins (3,29,30). This is a clear example for the need of care in the interpretation, because the reduced line broadening on TFP addition was initially interpreted as a competition between Ca2+ and TFP (30). It was later shown that the reduced line broadening is a result of a reduced exchange rate between free and bound Ca2+ -ions (29). This was shown quite elegantly with the use of shift reagents, whereby the 43Ca NMR resonances from free and protein bound ions could be shifted apart, Fig. 5.
226 Drakenberg Fig. 5. 43 Ca NMR spectra at 24.3 MHz obtained for a sample containing 1 mM bovine testis calmodulin, 100 mM Na ClO 4 at pH 7.0 with the following additions (A) 2.0 equ. Ca 2+ ; (B) 6 equ. Ca 2+ ; (C) 6 equ. Ca 2+ and 1.2 mM Dy(PPP) 2 7– ; (D) 6 mM Ca 2+ , 1.2 mM Dy(PPP) 2 7– , and 3 mM TFP. Reproduced with permission from ref. 29. As aforementioned, the 43 Ca NMR spectrum will depend strongly on the rate of calcium exchange between free calcium ions and ions bound to a protein. When the temperature dependence of the line shape is followed as a function of temperature, information about the exchange can be obtained if the exchange rate is faster than the relaxation rate of the free ion and not much
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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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Page 197 and 198: 178 Shaw 2.3. Other 1. Chemicals fo
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Page 203 and 204: 184 Brokx and Vogel Table 1 An Over
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Page 207 and 208: 188 Brokx and Vogel Fig. 2. 20% SDS
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Page 215 and 216: 196 Berliner Fig. 1. Aqueous X-band
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Page 221 and 222: 202 Berliner Fig. 5. Low-temperatur
Page 223 and 224: 204 Berliner 14. Musci, G., Reed, G
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
Page 237 and 238: 218 Drakenberg sands, of Hertz broa
Page 239 and 240: 220 Drakenberg 1. Bloch equations m
Page 241 and 242: 222 Drakenberg Fig. 2. Measurement
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Page 251 and 252: 232 Weljie and Heringa Table 1 Amin
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Page 255 and 256: 236 Weljie and Heringa diction meth
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Page 265 and 266: 246 Weljie and Heringa much larger
Page 267 and 268: 248 Weljie and Heringa ment require
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
Page 277 and 278: 258 Li et al. 7. Mineral Mixture (s
Page 279 and 280: 260 Li et al. 3. When the 45% D 2O/
Page 281 and 282: 262 Li et al. sion system works eff
Page 283 and 284: 264 Li et al. reliably produce high
Page 287 and 288: 268 Mal et al. NMR data, X-PLOR (11
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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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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METHODS IN MOLECULAR BIOLOGY TM •
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