Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

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Proteolytic Fragments of Ca 2+ -<strong>Binding</strong> <strong>Protein</strong>s 185 with trypsin or thrombin in our laboratory. The procedures are virtually identical for the highly homologous protein TnC. 2. Materials 2.1. Proteolytic Digestion 1. Lyophilized protein sample. Approximately 100 mg of CaM or TnC is needed for preparation of proteolytic fragments. It need not be totally pure of other polypeptides because the digest will be repurified anyway, but it should be free of salts, buffers, and other contaminants. 2. Buffers/reagents. Stock solutions that should be on hand include: a. 1 M Tris-HCl. Can be kept for months at 4°C. Generally, a pH 7.5 stock solution is kept on hand, but the pH can be altered when using it to make other buffers. b. 0.5 M Ethylenediamine tetraacetic acid (EDTA) or ethylene glycol bis- (β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), pH 8.0. Dissolve the free acid or the disodium salt and adjust the pH to 8.0 with solid NaOH and 12 M NaOH. Either solution is suitable; EDTA is a more generally used laboratory reagent, but EGTA is more appropriate because it is a more specific chelator for Ca2+ ions. These solutions can be kept indefinitely at room temperature. c. 0.1 M Dithiothreitol (DTT). Store frozen in small aliquots. d. 50 mM Ammonium bicarbonate (NH4HCO3). Store at 4°C. The pH of this solution at 4°C is 7.9. 3. Incubator (37°C). One used for growing bacterial plates is suitable. 4. UV/visible spectrophotometer. Used for checking protein concentrations in column fractions. See Fig. 1 for examples of the UV absorption spectra of CaM and its proteolytic fragments. 5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) apparatus. Used for checking purity of protein fractions. A 20% acrylamide gel is suitable for fragments of these sizes. See Fig. 2 for an example of an SDS-PAGE gel of CaM and its proteolytic fragments. 6. Chelex-100 matrix (Bio-Rad). Used for removal of metal ions from buffers and protein samples. 2.2. Tryptic Digestion Prepare all buffers fresh. 1. 1 mM HCl. 2. Buffer A: 50 mM NH 4HCO 3, 50 mM NaCl, pH 7.9. 3. Buffer B: 50 mM Tris-HCl, 1 mM CaCl 2, pH 7.5. 4. Buffer C: 2 mM Tris-HCl, 1 mM CaCl 2, pH 7.5. 5. Double-distilled (dd) H 2O, pH 7.5. Adjust pH very carefully with small amounts of NaOH and HCl.
186 Brokx and Vogel Fig. 1. UV absorption spectra of calmodulin and its proteolytic fragements. (A) calmodulin. (B) TR1C. (C) TR2C. 6. Trypsin (Sigma), from bovine pancreas; 10,000–13,000 BAEE U/mg protein. Lyophilized. Should be purchased TPCK-treated to reduce chymotrypsin activity (< 0.1 BTEE U/mg protein). Dissolve 5 mg in 1 mL 1 mM HCl. Prepare fresh. 7. Soybean trypsin inhibitor (STI) (Sigma). Lyophilized. 1 mg of STI inhibits 1–3 mg of trypsin with an activity of approx 10,000 BAEE units/mg protein. Dissolve 5 mg in 1 mL 1 mM HCl. Prepare fresh. 8. Sephadex G-50 (Pharmacia) (100 cm × 1.5 cm) equilibrated with buffer A. 9. Phenyl-Sepharose (Pharmacia) (approx 30 mL) equilibrated with buffer B.
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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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Page 227 and 228: 208 Clarke and Vogel Fig. 1. 113 Cd
Page 233 and 234: 214 Clarke and Vogel The linewidth
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
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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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