Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

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Gene Expression in Transfected Cells 359 11. Using a sterile Pasteur pipet, gently transfer all of the 0.5-mL transfected cells, including a white aggregate of cells that forms as a result of the electroporation, to the cell-culture flask containing 10 mL prewarmed medium from step 1. Be sure to transfer all of the cells by rinsing the cuvet with medium. 12. Incubate the transfected cells in the 37°C carbon dioxide incubator for 8–72 h (see Note 7). During the course of a long incubation, fast-growing cells may have to be diluted to prevent the culture from becoming too dense. 3.2. Lysis of the Cells 1. Harvest the 10.5 mL of transfected cells from Subheading 3.1., step 12 in 15-mL tubes by centrifugation at 250g for 10 min at room temperature. 2. Remove the supernatant. Resuspend the cells in 1 mL PBS and transfer them to 1.5-mL tubes. Centrifuge at 250g for 10 min at room temperature. 3. Remove the supernatant and resuspend the cell pellet in 100 µL of lysis buffer by gentle pipetting. Incubate for 5 min at room temperature. At this stage, the cell lysates can be stored at –70°C, or alternatively proceed directly to the assays. 3.3. β-Galactosidase and Luciferase Assays Centrifuge the lysed cells from Subheading 3.2., step 3 at 18,000g for 1 min at room temperature to pellet the cell debris. Place the tubes on ice and keep them on ice for the rest of the analysis. In order to compare different transfections with each other, the enzyme activity present in the same volume of lysate from each transfection has to be compared. However, the β-galactosidase can be measured from one volume and the luciferase from a different volume. 3.3.1. β-Galactosidase Assay 1. β-galactosidase catalyses the hydrolysis of ONPG to o-nitrophenol, which is yellow. Aliquot 250 µL ONPG solution (0.8 mg/mL in β-galactosidase assay buffer) to 1.5-mL tubes, preparing one tube more than the number of transfections. 2. Add 20 µL of the supernatant of each centrifuged lysate to a tube with ONPG solution. Mix well. 3. Incubate at room temperature until the samples are light yellow. Depending on the cell line, this will take anywhere from 5 min to overnight. If no yellow color appears overnight, your transfection has been unsuccessful. If this is the case, repeat Subheading 3.1. with different conditions (see Notes 3–6). 4. To directly compare the amount of β-galactosidase activity in each sample, stop the reactions of all samples after the same incubation time. When yellow color is reached in all samples, stop the reactions by adding 250 µL of 1 M Na 2CO 3. Also add 250 µL of 1 M Na 2CO 3 to the extra “blank” tube prepared in step 1. Mix well. 5. Measure the optical density at 420 nm (OD 420) of the reactions vs the “blank.” The β-galactosidase-catalyzed reaction is linear and can be accurately measured between an OD 420 of 0.2 and 0.8. If the OD 420 of your samples lie outside of this
360 Hughes et al. range, repeat steps 1–4 and leave the reactions for a longer time or analyze a smaller volume of each lysate (see Note 8). 3.3.2. Luciferase Assay Luciferase catalyzes the oxidation of its substrate luciferin, which leads to an emission of light. To carry out this reaction, use a commercially available kit (see Subheading 2.3.). The following protocol is a general outline of the procedure, but refer to the protocol that comes with your kit for specific details. 1. Prepare enough luciferase assay reagent for your samples. Equilibrate this to room temperature, the optimal temperature for luciferase activity measurements. 2. Aliquot 20 µL of the supernatant of each centrifuged lysate to a luminometer cuvet and leave at room temperature for 5 min to equilibrate. 3. Taking one cuvet at a time, add the recommended volume of luciferase assay reagent. Immediately measure the light emission of the reaction by placing the cuvet in a luminometer. It is important to transfer the cuvet to the luminometer as soon as possible after adding the reagent because the light intensity of the reaction is constant for only a few seconds (specified in your kit information) and then begins to decay. 3.4. Analysis of Data The luciferase activity value is a measure of transcription initiated from the reporter plasmid. However, the luciferase value of one transfection cannot without precaution be directly compared with the luciferase value of another, because the cells might not have received an identical amount of DNA during the electroporation. The β-galactosidase value is a measure of the efficiency of an individual transfection. Therefore, to compare different transfections, normalize the luciferase value of each individual transfection by dividing it by its corresponding β-galactosidase value. To determine the effect of (over-) expressing the cDNA, compare the luciferase/β-galactosidase value of that transfection to the luciferase/β-galactosidase value of cells transfected with an empty expression plasmid. 4. Notes 1. When choosing the promoters of the expression and internal control plasmids, consider the possibility that the protein you wish to (over-) express may influence transcription from the promoter. It is best to avoid such a promoter. 2. Although many electroporation cuvets are recommended for single use only, they can be reused up to 10 times with little effect on transfection efficiency. After use, cuvets are thoroughly rinsed in water and stored dry. Sterilize them by standing in a beaker of 70% ethanol a few hours before use, and then allow them to air dry (approx 30 min) in the hood prior to adding cells.
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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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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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Page 331 and 332: 312 Boyd et al. 2. When studying mu
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Page 337 and 338: 318 Yap et al. image representation
Page 339 and 340: 320 Yap et al. modified EF-hand is
Page 341 and 342: 322 Table 1 Angle and Distance Outp
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Page 345 and 346: 326 Kobayashi that S-100A1 and S-10
Page 347 and 348: 328 Kobayashi 3.2. Coupling of Crom
Page 349 and 350: 330 Kobayashi Fig. 2. Tricine/SDS/P
Page 351 and 352: 332 Kobayashi 0.1% TFA at a flow ra
Page 353 and 354: 334 Kobayashi Both recombinant S-10
Page 355 and 356: 336 Kobayashi Fig. 6. Affinity chro
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Page 359 and 360: 340 Walsh et al. verts CaM from an
Page 361 and 362: 342 Walsh et al. 11. Bovine serum a
Page 363 and 364: 344 Walsh et al. Fig. 1. CaM-depend
Page 367 and 368: 348 Walsh et al. 3.5. NOS Reduction
Page 373 and 374: 354 Walsh et al. 3. Cho, M. J., Vag
Page 375 and 376: 356 Hughes et al. The electroporati
Page 377: 358 Hughes et al. 4. 1 M Na 2CO 3.
Page 381 and 382: 362 Hughes et al. Table 1 Examples
Page 383 and 384: 20 Dean, Kelsey, and Reik
Page 385 and 386: 366 Persechini of the different CaM
Page 387 and 388: 368 Persechini Fig. 1. Schematic re
Page 389 and 390: 370 Persechini 2. 1 L of Terrific b
Page 391 and 392: 372 Persechini Fig. 4. Titration wi
Page 393 and 394: 374 Persechini Table 1 Parameters f
Page 395 and 396: 376 Persechini Fig. 6. The [Ca 2+ -
Page 397 and 398: 378 Persechini determined if the di
Page 399 and 400: 380 Persechini relatively insensiti
Page 401 and 402: 382 Persechini 18. Chafouleas, J. G
Page 403 and 404: 384 Török et al. actions in the c
Page 405 and 406: 386 Török et al. 3. The reaction
Page 407 and 408: 388 Török et al. Fig. 2. Electros
Page 409 and 410: 390 Török et al. Fig. 3. Peptides
Page 411 and 412: 392 Török et al. Table 1 Peptide
Page 413 and 414: 394 Török et al. Fig. 7. Nanospra
Page 415 and 416: 396 Török et al. Fig. 9. Nanospra
Page 417 and 418: 398 Török et al. Fig. 11. Electro
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Page 421 and 422: 402 Török et al. Fig. 14. Localiz
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Page 425 and 426: 406 Török et al. the significantl
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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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