Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

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Steady-State Fluorescence Spectroscopy 83 acrylamide. KI is sensitive to light degradation; therefore, solutions should be made fresh and care taken with handling. 3. Alternatively, the sample can be exchanged from a different buffer into the fluorescence buffer by passing the sample through a size exclusion column containing G-25 resin (Pharmacia). 4. There are also several sites on the World Wide Web where one can obtain estimates of protein extinction coefficients using more complex factors. For example, the ProtParam tool estimates ε 280 based on both fully reduced cys residues, or on complete disulphide bond formation, in addition to tryptophan and tyrosine contributions. 5. In preparation of samples, the following concentration guidelines from our lab might be useful. Protein samples containing the fluorophore of interest are used at a concentration of 10–12 µM. Any other protein species binding to this one is added to a 10–20% molar excess. For addition of Ca 2+ ions, a 50-mM stock of CaCl 2 is added to a final concentration of 1 mM. Calcium-free studies are ensured by the addition of a 500 mM stock of EDTA to a final concentration of 5 mM. 6. The optimal bandpass settings are dependent on numerous technical factors including the optics of the instrument, the signal to noise ratio of the photomultiplier tube, and the signal from the sample, all of which is unique to a given setup. Generally speaking, as one increases the excitation/emission bandpass sizes, more electronic transitions are excited/detected simultaneously; hence, fine structure is lost at larger values. It is important to note that the bandpass value is taken as the full slitwidth, with the chosen wavelength at the center. For example, a bandpass of 5 nm for excitation at 295 implies light from 292.5 to 297.5 nm would pass through, unlike the definition used in other spectroscopic methods (i.e., 295 +/– 5 nm). If the intensity of the fluorescence signal being monitored shows small changes with mediocre signal to noise, the emission bandpass can be increased (to 50 nm for example) in order to provide the equivalent of an integral of the emission peak. 7. Several methods of referencing can be used for normalization and calibration of the fluorimeter. The best trade-off between simplicity and accuracy is to use a chemical sample of known composition and concentration (such as 5 µM tryptophan at pH 7.0) before each fluorescence session and normalize the acquired data to this reference. 8. An ethanol or detergent wash followed by a minimum of 10 rinsings with distilled water should be performed between samples. For smaller cells, an air aspirator will facilitate the cleaning and drying process. 4.2. Other Fluorescence Methods and Applications 9. After having reviewed the literature and performed initial fluorescence experiments on a (suspected) calcium-binding protein, here are several simple experiments suggested for initial analysis: a. Excitation and emission spectra of protein alone, and then +/– Ca2+ titration.
84 Table 1 Advanced Fluorescence Methods and Selected Applications Protein/ Fluorophore Brief method details Application Ref. Parvalbumin, oncomodulin, calmodulin/Tb 3+ Calmodulin, troponin C/tryptophan, DANSYL Calmodulin/tryptophan Calmodulin/tyrosine 68Calmodulin/GFP Phospholipid scramblase/tryptophan, Tb 3+ C2 domain of Cytosolic Phospholipase A 2/fluorescin Annexin V/fluorescin, NBD FRET from aromatic amino acids in the Ca 2+ - binding loops of the proteins to bound Tb 3+ . Frequency domain FRET used to probe the distance between the DANSYLated N-terminus and Trp of melittin. These measurements were performed for the free peptide as well as in complexes with CaM, TnC, and in vesicles. Steady state fluorescence of CaM complexes with CaM-target peptides. CaM proteins were engineered such that methionine residues were replaced with unnatural amino acid analogs. FRET between two tyrosines by using a nitro-tyrosine derivative as the acceptor, and a normal tyrosine as the donor. Distinctly colored mutant Green Fluorescent proteins are genetically appended to the termini of a CaM/CaM-target chimera and monitored for fluorescence resonance energy transfer. FRET evidence that there is coordinate metal ion binding from protein trp to Tb 3+ in an EF- hand-like domain. The Tb 3+ is competed out by Ca 2+ . Cysteine scanning mutagenesis used to attach a fluorescin probe in 16 locations. Ca 2+ triggered environmental changes were probed based on intensity changes and Stern- Volmer quenching constants. Fluorescence recovery after photobleaching experiments employed to probe the concentration effects of annexin V on its lateral mobility in a mixed lipid system. Use of Tb 3+ as a sensitive luminescent probe of the structure and function of EF-hand Ca 2+ - binding loops Determine the distribution of distances present in solution between the two fluorophores. The result can be interpreted as the degree of conformational freedom. Probing the role of methionine side-chains in the sequestering of CaM-target peptides. Determination of distance separation in both the apo and Ca 2+ -saturated states. Detection of localized concentrations of Ca 2+ . Can be targeted to specific organelles within living cells. Confirmation that the EF-hand looplike segement contributes directly to Ca 2+ - binding. Indication that the membrane docking surface of the C2 domain is localized to the same surface that binds a pair of Ca 2+ ions. Demonstration that the annexin is hindered by its specific interaction with one type of lipid; localization of this interaction to the headgroup. (15) (16) (10,12) (17) (18) (19) (20) (21) 84 Weljie and Vogel
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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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Page 61 and 62: 42 Haiech and Kilhoffer 62. Becking
Page 63 and 64: 44 Martin and Bayley Fig. 1. Absorp
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Page 67 and 68: 48 Martin and Bayley 4. Set the tem
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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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Page 105 and 106: 86 Weljie and Vogel References 1. L
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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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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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METHODS IN MOLECULAR BIOLOGY TM •
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