Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

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Calcium Binding to Proteins 19 K 1–K N (where N is the number of sites that are strong enough to compete with the chelator) are obtained by nonlinear least squares fitting to the absorbance as a function of total calcium concentration. An analysis based on concentration (not activities) can be performed as follows (see Note 10). The total Ca 2+ -concentration at each titration point i (CATOT i), is calculated from the initial (see Note 11) and added Ca 2+ .A nominal value for the protein concentration at each titration point (CP i) is calculated from the initial protein concentration based on the weight of the lyophilized protein. CATOT i and CP i are adjusted for the dilution imposed by the calcium additions, as is CQ i, the chelator concentration at titration point i. Fixed parameters in the fit are KDQ, CQ i, CP i, and CATOT i. KDQ is the Ca 2+ -dissociation constant of the chelator. Variable parameters in the fit are K 1–K N, AMAX, AMIN, and F. AMAX and AMIN are the absorbances that the initial (nondiluted) solution would have had if it was completely Ca 2+ -free or contained saturating amounts of Ca 2+ , respectively. F is a correction factor that accounts for the fact that the protein concentration obtained by weight can be off by 10–20% because of residual water in lyophilized protein and because of errors in weight caused by the small (0.7–1.5 mg) quantities used (see Note 12). For each set of values of the variable parameters, the Newton-Raphson method is used to solve the free Ca 2+ concentration, Y, at each titration point, i, from the following equation: F·CPi ∑ k N CQi·Y (kYk· Kj) =l j =l Y = CATOTi – ———— – ———————— (3) Y + KDQ 1 + ∑ k N (Yk· ∏ =l j k Kj) =l which states that the free Ca2+ equals the total Ca2+ subtracted by the chelatorbound Ca2+ and the protein-bound Ca2+ . The absorbance at point i is calculated as Y CQi Acalculated,i = AMAX – (AMAX – AMIN) [ ·————] · ——— (4) Y + KDQ Ql where CQ 1 is the initial chelator concentration. Thus the changes in absorbance are assumed to arise from the chelator only. The sum of the squares of residuals (or error square sum) χ 2 , is obtained by summing over all points in the titration χ 2 = ∑ (A calculated,i – A measured,i) 2 (5) The variable parameters are iterated in a separate procedure until an optimal fit (minimum χ 2 ) is found. Start with initial guesses at both sides of the parameter values of best fit, to make sure that the same result is obtained. To estimate the errors in the parameter values, one may fix one parameter, for example K 1, and ∏ k
20 Linse iterate the other parameters to obtain an optimal fit. Then fix K 1 at a new value and fit again. Repeat until you have found the values of K 1 that lead to a doubling of χ 2 . In general, AMAX, AMIN, and F are better determined than the binding constants (see Note 13). If the protein binds calcium with positive cooperativity (see Note 14), the product of the binding constants is better determined than the individual constants. 3.3. Stoichiometry of Calcium Binding The chelator method can be used to measure the stoichiometry of calcium binding. For such applications, extra care has to be taken to measure the protein concentration of the titrand and its initial and final calcium concentration. 1. Dissolve the protein in 3 mL chelator solution to approx 30 µM. 2. Withdraw 200 µL. Freeze dry for acid hydrolysis. 3. Use 2.5 mL as titrand. 4. Save the rest for atomic absorption spectroscopy for initial calcium concentration analysis. 5. Record A 263 for the titrand. 6. Add a calcium aliquot to the titrand and mix. Record A 263. 7. Repeat step 6 until no significant A 263 change has been observed over the last five points. 8. Withdraw an aliquot of the titrated titrand for atomic absorption spectroscopy for calcium analysis. 9. In the computer fitting, set the initial protein concentration to the value obtained from the amino acid analysis, and use a fixed factor F = 1.0. The number of macroscopic binding constants needed to obtain an optimal fit will be the same as the number of sites with affinities of similar value as the chelator. The initial calcium concentration used in the fit is obtained from the analysis at step 4. Check that the total calcium concentration at the last titration point is equal to the value obtained from the analysis at step 8. 3.4. Examples of Titration Data Examples of experimental data and fitted curves are shown in Fig. 2. In the absence of calcium binding to the protein, the absorbance will decrease linearly until the total calcium concentration equals the chelator concentration. A linear decrease will be seen also when the protein has a site with the same Ca2+ affinity as the chelator, but more calcium will be needed to saturate the chelator. If the protein binds calcium weaker or stronger than the chelator, the binding curve will be no longer be a straight line, but will bend in a different direction depending on whether the affinity for the protein is higher or lower than for the chelator (see Fig. 2A). Examples of experimental data for proteins with one, two, or three high-affinity calcium-binding sites are shown in Fig. 2B. When the protein binds calcium at more than one site in a sequential manner
Page 1 and 2: Methods in Molecular BiologyTM Biol
Page 3 and 4: M E T H O D S I N M O L E C U L A R
Page 5 and 6: © 2002 Humana Press Inc. 999 River
Page 8 and 9: Preface Calcium plays an important
Page 10 and 11: Contents Dedication ...............
Page 12: Contents xi 26 Enzymatic Assays to
Page 15 and 16: xiv Contents of Companion Volume 15
Page 17 and 18: xvi Contributors LESLIE D. HICKS
Page 19 and 20: 20 Dean, Kelsey, and Re
Page 21 and 22: 2 Yazawa
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Page 27 and 28: 8 Yazawa DuPont-NEN and the molar c
Page 29 and 30: 10 Yazawa Fig. 4. Examples of the C
Page 31 and 32: 12 Yazawa 5. Plot the calculated Ca
Page 33 and 34: 14 Yazawa 12. Starovasnik, M. A., D
Page 35 and 36: 16 Fig. 1. Molecular structures and
Page 37: 18 Linse 7. 0.1 M EDTA. Dissolve 37
Page 41 and 42: 22 Linse tive to small alterations
Page 43 and 44: 24 Linse References 1. Tsien, R. Y.
Page 45 and 46: 26 Haiech and Kilhoffer to use the
Page 47 and 48: 28 Haiech and Kilhoffer where γ is
Page 49 and 50: 30 Haiech and Kilhoffer reporter gr
Page 51 and 52: 32 Haiech and Kilhoffer tein with i
Page 53 and 54: 34 Haiech and Kilhoffer Calmodulin
Page 55 and 56: 36 Haiech and Kilhoffer Fig. 3. Mec
Page 57 and 58: 38 Haiech and Kilhoffer We may sugg
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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
Page 65 and 66: 46 Martin and Bayley evaporation. C
Page 67 and 68: 48 Martin and Bayley 4. Set the tem
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Page 71 and 72: 52 Martin and Bayley discussed by M
Page 73 and 74: 54 Martin and Bayley References 1.
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Page 81 and 82: 62 Fabian and Vogel perature, ionic
Page 83 and 84: 64 Fabian and Vogel Fig. 3. IR spec
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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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20 Dean, Kelsey, and Reik
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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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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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