Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

More documents

Recommendations

Info

Monitoring Ca 2+ -Calmodulin Concentration 369 Fig. 2. Maps of vectors for expression of indicators in bacterial (upper map) or mammalian (lower map) cells. The positions of features and key restriction sites are indicated. of these difficult variants, but we have found it to give reliable yields of all GFP-based indicators. 1. 50 mL of Terrific broth is inoculated with BL21(DE3) cells transformed with the desired plasmid, and the cells are grown under 50 µg/mL kanamycin selection overnight at 37°C in a shaker running at 300 rpm.
370 Persechini 2. 1 L of Terrific broth in a 2800-mL baffled flask (Bellco Glass, Inc., Vineland, NJ) is inoculated with the 50-mL overnight culture. It is not necessary to include kanamycin at this stage. Cells are incubated with shaking at 23°C for 6 h or until the culture has reached an OD 660 of 0.6–0.8, and expression is induced by adding 0.5 mM IPTG. Incubation at 23°C is continued for approx 40 h. 3. The bacterial cells are pelleted at 5000 rpm for 10 min in a Sorvall GS-3 rotor at 4°C, and the cell pellet is resuspended in 100 mL of 50 mM Tris-HCl; pH 8.0. The centrifugation step is repeated, and the cell pellet is resuspended in 100 mL of resuspension buffer. 4. 200 µg/mL egg-white lysozyme is added to the cell suspension and it is immediately transferred to centrifuge tubes and incubated on ice for 30 min. 5. The cells are then sonicated for 2 min using a Branson Sonifier 450 with microtip attachment. We use a 40% duty cycle and the maximum microtip power output. The lysed cells are then subject to centrifugation for 20 min at 10,000 rpm in a Sorvall SS-34 rotor at 4°C. 6. DNase buffer is added to the supernatant fraction, followed by centrifugation at 25,000 rpm for 60 min in a Beckman SW27 rotor at 4°C. DNase I is included so that nucleic acids do not clog the affinity column during the final purification step. The supernatant fraction from this procedure is suitable for a preliminary characterization of the indicator fluorescence, and culture volumes can be reduced by a factor of at 20–30 if only the crude supernatant fraction is required. 7. A 1-mL Pharmacia (Uppsala, Sweden) HiTrap ® metal chelating column is washed with 5 mL water, primed with 0.5 mL of 0.1 M NiCl, and washed with an additional 5 mL water. The supernatant fraction is then pumped onto the column. A greenish-yellow accumulation of bound indicator on the column should develop during this process. 8. The column is washed with 25 mL of column buffer “a,” followed by 25 mL column buffer “b,” and 15 mL of column buffer “c.” 9. The bound indicator is eluted into a minimal volume of elution buffer and dialyzed exhaustively against dialysis buffer. 10. The concentration of indicators constructed with the ECFP/EYFP FRET pair are determined by optical absorbance using a ε 513 of 89 mM/cm. We no longer routinely perform a gel electrophoretic analysis of the final product, but in the past have consistently observed a purity level ≥ 80%, with the highest purity observed when the affinity column is saturated with 6-His-tagged protein prior to elution, which seems to discourage nonspecific binding. After dialysis, indicator proteins can be stored frozen at –80 °C for several months. 3.1.2. Spectral Characterization and K d Determination For each indicator, it is important at least to determine the changes in the excitation and emission spectra that occur upon binding Ca 2+ –CaM, and the apparent dissociation constant for Ca 2+ –CaM. It is also important to verify the Ca 2+ -dependence of its interaction with CaM. All in vitro fluorescence measurements are performed using a Photon Technologies International
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
Page 23 and 24:
4 Yazawa Fig. 1. Schematic represen
Page 25 and 26:
6 Yazawa Fig. 2. Shop drawing for t
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 and 38:
18 Linse 7. 0.1 M EDTA. Dissolve 37
Page 39 and 40:
20 Linse iterate the other paramete
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
Page 59 and 60:
40 Haiech and Kilhoffer 29. Stewart
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
Page 69 and 70:
50 Martin and Bayley Fig. 2. Near-U
Page 71 and 72:
52 Martin and Bayley discussed by M
Page 73 and 74:
54 Martin and Bayley References 1.
Page 75 and 76:
20 Dean, Kelsey, and Reik
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
Page 91 and 92:
72 Fabian and Vogel 4. ATR-FTIR spe
Page 93 and 94:
74 Fabian and Vogel 25. Georg, H.,
Page 95 and 96:
76 Weljie and Vogel Fig. 1. Simplif
Page 97 and 98:
78 Weljie and Vogel Fig. 3. Steady-
Page 99 and 100:
80 Weljie and Vogel VIS spectrophot
Page 101 and 102:
82 Weljie and Vogel 4. A series of
Page 103 and 104:
84 Table 1 Advanced Fluorescence Me
Page 105 and 106:
86 Weljie and Vogel References 1. L
Page 107 and 108:
Page 109 and 110:
90 Johnson and Tikunova is removed
Page 111 and 112:
92 Johnson and Tikunova function of
Page 113 and 114:
94 Johnson and Tikunova Fig. 2. Rat
Page 115 and 116:
96 Johnson and Tikunova Fig. 3. Rat
Page 117 and 118:
98 Johnson and Tikunova Fig. 4. Ca
Page 119 and 120:
100 Johnson and Tikunova cence inte
Page 121 and 122:
102 Johnson and Tikunova 6. Johnson
Page 123 and 124:
104 Julenius Fig. 1. Under conditio
Page 125 and 126:
106 Julenius 3. Mix 50 µL of NHS s
Page 127 and 128:
108 Julenius Fig. 3. A typical sens
Page 129 and 130:
110 Julenius (2), is used in the Ia
Page 131 and 132:
Page 133 and 134:
114 Lopez and Makhatadze Fig. 1. Th
Page 135 and 136:
116 Lopez and Makhatadze 6. Buffers
Page 137 and 138:
118 Lopez and Makhatadze The excess
Page 139 and 140:
Page 141 and 142:
122 Lopez and Makhatadze Fig. 1. Is
Page 143 and 144:
124 Lopez and Makhatadze 2.5 mL are
Page 145 and 146:
126 Lopez and Makhatadze pendence o
Page 147 and 148:
128 Hicks et al. equipped with a pu
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
Page 153 and 154:
134 Hicks et al. Fig. 3. Sedimentat
Page 155 and 156:
136 Hicks et al. 5. Hayes, D. B. (M
Page 157 and 158:
138 Trewhella and Krueger This chap
Page 159 and 160:
140 Trewhella and Krueger of the sc
Page 161 and 162:
142 Trewhella and Krueger Table 1 C
Page 163 and 164:
144 Trewhella and Krueger beam, or
Page 165 and 166:
146 Trewhella and Krueger At very l
Page 167 and 168:
148 Trewhella and Krueger analytica
Page 169 and 170:
150 Trewhella and Krueger collapsed
Page 171 and 172:
152 Trewhella and Krueger P(r) func
Page 173 and 174:
154 Trewhella and Krueger Fig. 3. S
Page 175 and 176:
156 Trewhella and Krueger concentra
Page 177 and 178:
158 Trewhella and Krueger by a Nati
Page 179 and 180:
Page 181 and 182:
162 Doherty-Kirby and Lajoie metal
Page 183 and 184:
164 Doherty-Kirby and Lajoie two Ca
Page 185 and 186:
166 Doherty-Kirby and Lajoie 7. Pep
Page 187 and 188:
168 Doherty-Kirby and Lajoie Fig. 1
Page 189 and 190:
170 Doherty-Kirby and Lajoie Fig. 2
Page 191 and 192:
172 Doherty-Kirby and Lajoie 5. Alt
Page 193 and 194:
174 Doherty-Kirby and Lajoie phosph
Page 195 and 196:
176 Shaw Fig. 1. Ribbon drawing of
Page 197 and 198:
178 Shaw 2.3. Other 1. Chemicals fo
Page 199 and 200:
180 Shaw 3.3.3. Purification 1. Con
Page 201 and 202:
182 Shaw 10. Hodges, R. S., Semchuk
Page 203 and 204:
184 Brokx and Vogel Table 1 An Over
Page 205 and 206:
186 Brokx and Vogel Fig. 1. UV abso
Page 207 and 208:
188 Brokx and Vogel Fig. 2. 20% SDS
Page 209 and 210:
190 Brokx and Vogel it through an a
Page 211 and 212:
192 Brokx and Vogel 8. Weljie, A. M
Page 213 and 214:
Page 215 and 216:
196 Berliner Fig. 1. Aqueous X-band
Page 217 and 218:
198 Berliner in the Bruker instrume
Page 219 and 220:
200 Berliner Fig. 3. ESR spectra of
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 229 and 230:
Page 231 and 232:
Page 233 and 234:
214 Clarke and Vogel The linewidth
Page 235 and 236:
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
Page 243 and 244:
224 Drakenberg Fig. 3. (A) 43 Ca NM
Page 245 and 246:
226 Drakenberg Fig. 5. 43 Ca NMR sp
Page 247 and 248:
228 Drakenberg NMR at sub-mM concen
Page 249 and 250:
230 Drakenberg 30. Shimizu, T., Hat
Page 251 and 252:
232 Weljie and Heringa Table 1 Amin
Page 253 and 254:
234 Weljie and Heringa Table 2 Webs
Page 255 and 256:
236 Weljie and Heringa diction meth
Page 257 and 258:
238 Weljie and Heringa these databa
Page 259 and 260:
240 Weljie and Heringa lineages. Th
Page 261 and 262:
242 Weljie and Heringa compared, an
Page 263 and 264:
244 Weljie and Heringa sequences as
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
Page 273 and 274:
254 Weljie and Heringa
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 285 and 286:
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
Page 295 and 296:
276 Mal et al. Assuming a rigid bod
Page 297 and 298:
278 Mal et al. assign (segid A and
Page 299 and 300:
280 Mal et al. References 1. Drenth
Page 301 and 302:
282 Mal et al. 33. Jeener, J., Meie
Page 303 and 304:
Page 305 and 306:
286 Werner et al. Our research has
Page 307 and 308:
288 Fig. 1. (A) T 1, T 2 and the he
Page 309 and 310:
290 Werner et al. tion, using gradi
Page 311 and 312:
292 Werner et al. Fig. 3. (A) Rotat
Page 313 and 314:
294 Werner et al. Fig. 4. (A) Line-
Page 315 and 316:
296 Werner et al. Table 2 Models of
Page 317 and 318:
298 Werner et al. 9. Reinhardt, D.
Page 319 and 320:
300 Werner et al. 39. Press, W. H.,
Page 321 and 322:
302 Boyd et al. utilize residual di
Page 323 and 324:
304 Boyd et al. Fig. 1. (A) The sol
Page 325 and 326:
306 Boyd et al. or significant peak
Page 327 and 328:
308 Boyd et al. Fig. 3. Histogram o
Page 329 and 330:
310 Boyd et al. total and NOE energ
Page 331 and 332:
312 Boyd et al. 2. When studying mu
Page 333 and 334:
314 Boyd et al. 4. Downing, A. K.,
Page 335 and 336:
316 Boyd et al. 35. Schulte-Herbrug
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
Page 343 and 344: 324 Yap et al. 2. Ikura, M. (1995)
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
Page 357 and 358: 20 Dean, Kelsey, and Reik
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 and 378: 358 Hughes et al. 4. 1 M Na 2CO 3.
Page 379 and 380: 360 Hughes et al. range, repeat ste
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: 368 Persechini Fig. 1. Schematic re
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
Page 419 and 420: 400 Török et al. 3 µM FL-calmodu
Page 421 and 422: 402 Török et al. Fig. 14. Localiz
Page 423 and 424: 404 Török et al. Fig. 16. Indicat
Page 425 and 426: 406 Török et al. the significantl
Page 427 and 428: 408 Török et al.
Page 429 and 430: 410 Index substitutes, see Fluoresc
Page 431 and 432: 412 Index Stern-Volmer plot, 78, 81
Page 433 and 434: 414 Index Phenothiazines, see Calmo
Page 435: METHODS IN MOLECULAR BIOLOGY TM •
show all

Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

Create successful ePaper yourself

Delete template?

Save as template?