Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

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Fluorescence Methods for Ca 2+ Exchange 101 chelators that undergo changes in absorption (or fluorescence) upon Ca 2+ -binding (as discussed by S. Linse in Chapter 2). 13. The rates of Ca 2+ dissociation from proteins can also be determined using the luminescent trivalent cation terbium (Tb 3+ ). Tb 3+ undergoes a large increase in fluorescence (an phosphorescence) upon binding to Ca 2+ -binding proteins. When Tb 3+ is reacted with proteins containing bound Ca 2+ (or Mg 2+ ), its luminescence increases at the rate of Ca 2+ (or Mg 2+ ) dissociation. We have used Tb 3+ fluorescence and stopped-flow methodology to determine the rates of Ca 2+ and Mg 2+ dissociation from parvalbumin and EGTA (16). Ca 2+ exchange rates can also be determined by NMR spectroscopy (see T. Drakenberg, Chapter 18). 14. We have recently introduced a method that allows the generation of Ca 2+ transients of various amplitudes and duration in a stopped-flow apparatus. This method is based on the fact that EGTA and Mg-EDTA have a slow Ca 2+ on-rate and when Ca 2+ is rapidly mixed with a solution containing EGTA (or Mg-EDTA), [Ca 2+ ] transiently rises until it is bound by chelator. This has allowed us to produce “artificial” Ca 2+ transients which vary in duration from 0.1 to 50 ms and to observe the transient activation of various Ca 2+ -binding proteins and Ca 2+ dependent enzymes in response to these transients (15). Thus, using stopped-flow techniques similar to those discussed above it is possible to produce Ca 2+ transients and follow the response of Ca 2+ -binding proteins to these transients in a stoppedflow apparatus. Acknowledgments This work was supported by a grant from the National Institutes of Health (DK33727). References 1. Johnson, J. D. and Potter J. D. (1978) Detection of two classes of calcium binding sites in troponin C with circular dichroism and tyrosine fluorescence. J. Biol. Chem. 253, 3775–3777. 2. Dedman, J. R., Potter, J. D., Jackson, R. L., Johnson, J. D., and Means, A. R. (1977) Physicochemical properties of rat testis calcium-dependent regulator protein of cyclic nucleotide phosphodiesterase. J. Biol. Chem. 252, 8415–8422. 3. George, S. E., Su, Z., Fan, D., and Means, A. R. (1993) Calmodulin-cardiac troponin C chimeras. Effects of domain exchange on calcium binding and enzyme activatin. J. Biol. Chem. 268, 25,213–25,220. 4. Pearlstone, J. R., Borgford, T., Chandra, M., Oikawa, K., Kay, C. M., Herzberg, O., Moult, J., Herklotz, A., Reinach, R. C., and Smillie, L. B. (1992) Construction and characterization of a spectral probe mutant of troponin C: application to analyses of mutants with increased calcium affinity. Biochemistry 31, 6545–6553. 5. Johnson, J. D., Collins, J. H., and Potter, J. D. (1978) Dansylaziridine-labeled Troponin C: a fluorescent probe of calcium binding to the calcium specific regulatory sites. J. Biol. Chem. 253, 6451–6458.
102 Johnson and Tikunova 6. Johnson, J. D., Collins, J. H., Robertson, S. P., and Potter, J. D. (1980) A fluorescent probe study of calcium binding to the calcium specific sites of cardiac troponin and troponin C. J. Biol. Chem. 255, 9635–9640. 7. Bayley, P., Ahlstrom, P., Martin, S. R., and Forsen, S. (1984) The kinetics of calcium binding to calmodulin: Quin 2 and ANS stopped-flow fluorescence studies. Biochem. Biophys. Res. Commun. 120, 185–191. 8. Martin, S. R., Maune, J. F., Beckingham, K., and Bayley, P. (1992) Stopped-flow studies of calcium dissociation from calcium-binding -site mutants of Drosophila melanogaster calmodulin. Eur. J. Biochem. 205, 1107–1114. 9. Robertson, S. and Potter, J. D. (1984) The regulation of free calcium ion concentration by metal chelators. Methods Pharmacol. 5, 63–75. 10. Fabiato, A. (1988) Computer programs for calculating total from specificed free or free from specifed total ionic concentrations in aqueous solutions containing multiple metal ligands. Methods Enzymol. 157, 378–417. 11. Schoenmakers, T. J., Visser, G. J., Flik, G., and Theuvenet, A. P. (1992) Chelator: an improved method for computing metal ion concentrations in physiological solutions. Biotechniques 6, 870–874. 12. Haugland, R. P. (1996) Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, (Spence, M. T. Z., ed.), 6th ed., Molecular Probes, Inc., Europe, UK, p. 505. 13. Johnson, J. D., Snyder, C., Walsh, M. P., and Flynn, M. (1996) Effects of myosin light chain kinase and peptides on calcium exchange with the N- and C-terminal calcium binding sites of calmodulin. J. Biol. Chem. 271, 761–767. 14. Brown, S. E., Martin, S. R., and Bayley, P. M. (1997) Kinetic control of the dissociation pathway of calmodulin-peptide complexes. J. Biol. Chem. 272, 3389–3397. 15. Davis, J. P., Tikunova, S. B., Walsh, M. P., and Johnson, J. D. (1999) Characterizing the response of calcium signal transducers to generated calcium transients. Biochemistry 38, 4235–4244. 16. Johnson, J. D., Jiang, Y. D., and Rall, J. A. (1999) Intracellular EDTA mimics parvalbumin in the promotion of skeletal muscle relaxation. Biophys. J. 76, 1514–1522.
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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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Page 77 and 78: 58 Fabian and Vogel are equipped wi
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
Page 89 and 90: 70 Fabian and Vogel Fig. 5. Amide I
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Page 103 and 104: 84 Table 1 Advanced Fluorescence Me
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Page 111 and 112: 92 Johnson and Tikunova function of
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Page 125 and 126: 106 Julenius 3. Mix 50 µL of NHS s
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Page 143 and 144: 124 Lopez and Makhatadze 2.5 mL are
Page 145 and 146: 126 Lopez and Makhatadze pendence o
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Page 149 and 150: 130 Hicks et al. Fig. 2. Debye plot
Page 151 and 152: 132 Hicks et al. and 1.0 mg/mL for
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Page 157 and 158: 138 Trewhella and Krueger This chap
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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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