Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

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Gene Expression in Transfected Cells 361 3. The optimal amount of expression plasmid depends on the cell type and the efficiency of the transfection and the expression of the plasmid in these cells. It might also depend on effects of the introduced plasmid or the expressed protein, for example if the protein becomes toxic to the cell above a certain concentration. Therefore try a range of DNA concentrations to determine the optimal concentration for your purposes. 4. The cells can alternatively be resuspended in PBS or PBS supplemented with 20 mM HEPES, pH 7.1, for the electroporation procedure. HEPES provides extra buffering capacity to reduce the pH change that occurs at the electrodes, a cause of cell death during electroporation. There appears to be little difference in transfection efficiency between PBS, PBS-HEPES, or culture medium, but cells may show enhanced survival when electroporated in culture medium. Transfection efficiency can be affected by the salt concentration of the electroporation buffer (12). Furthermore, addition of carrier DNA can improve transfection efficiency (12). It is therefore recommended to use carrier DNA, such as an inert plasmid, when the amount of expression plasmid is low. 5. For cells more resistant to transfection, efficiency may be improved by incubating the cells (in the cuvets) on ice for 5 min before and after electroporation. This may reduce the kinetics of pore closure and thus provide more time for the DNA to enter the cell. It may also protect the cells from heat damage when subjected to the electric pulse. 6. The voltage setting is the most critical parameter of the transfection procedure. Too low a voltage will have no effect on the cell membrane, but too high a voltage irreversibly damages the cell. The optimal voltage should be determined empirically using your experimental conditions. As a guideline, Table 1 lists voltages that are successful for a variety of cell lines using the indicated electroporation system. Comprehensive studies of optimal voltages for other cell types appear in the literature (for example, refs. 4,12–14). 7. Transfected cells can be analyzed several hours to several days after electroporation. Times shorter than 8 h are usually not long enough for reasonable expression of the protein. Over a period of several days, the cells lose transfected DNA and thus expression from the plasmid will gradually decrease. For studies of expression over a longer period of time, stable transfectants are needed. Albeit at a very low frequency, exogenously introduced DNA can insert into the chromosomal DNA. If the transfected expression plasmid also encodes a selectable marker, for example a drug resistance gene, it is possible to select for and amplify these cells (15–17). These stable transfectants can continue to express the integrated gene for an indefinite time. 8. If the β-galactosidase activity of the samples varies more than fourfold, it is not possible to stop the reactions when all samples lie in the OD 420 range of 0.2–0.8. In this case, stop individual reactions when they have reached the appropriate color, record the reaction time, and wait until all reactions are finished before measuring the OD 420 of the samples. The ONPG hydrolysis per time unit is then calculated.
362 Hughes et al. Table 1 Examples of Suitable Voltages for Electroporation Using the Gene Pulser ® II Electroporation System (Bio-Rad) with a Capacitance of 950 µF and a Time Constant of 15–20 ms Cell line Voltage Cell line Voltage MOLT-4 290 Jurkat 330 DG-75 300 K-562 340 HL-60 300 Primary B-cells 340 Raji 300 CTLL-2 360 BL-41 320 EL4 370 Acknowledgments This work was supported by grants from the Swedish Cancer Society, the Swedish Research Council for Engineering Sciences/SSF, and the Swedish Natural Science Research Council. References 1. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Expression of cloned genes in cultured mammalian cells, in Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp. 16.1–16.81. 2. Keown, W. A., Campbell, C. R., and Kucherlapati, R. S. (1990) Methods for introducing DNA into mammalian cells. Methods Enzymol. 185, 527–537. 3. Spector, D. L., Goldman, R. D., and Leinwand, L. A., eds. (1998) Preparation of macromolecules and introduction into cells, in Cells: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp. 82.1–93.22. 4. Potter, H. (1988) Electroporation in biology: methods, applications, and instrumentation. Anal. Biochem. 174, 361–373. 5. Tsong, T. Y. (1991) Electroporation of cell membranes. Biophys. J. 60, 297–306. 6. Rasmussen, C. D., Means, R. L., Lu, K. P., May, G. S., and Means, A. R. (1990) Characterization and expression of the unique calmodulin gene of Aspergillus nidulans. J. Biol. Chem. 265, 13,767–13,775. 7. Colomer, J., Agell, N., Engel, P., and Bachs, O. (1994) Expression of calmodulin and calmodulin binding proteins in lymphoblastoid cells. J. Cell Physiol. 159, 542–550. 8. Ye, Q., Wei, Y., Fischer, R., Borner, C., and Berchtold, M. W. (1997) Expression of calmodulin and calmodulin binding proteins in rat fibroblasts stably transfected with protein kinase C and oncogenes. Biochim. Biophys. Acta 1359, 89–96. 9. Zheng, Q. and Chang, D. C. (1991) High-efficiency gene transfection by in situ electroporation of cultured cells. Biochim. Biophys. Acta 1088, 104–110. 10. Raptis, L. H., Firth, K. L., Brownell, H. L., Todd, A., Simon, W. C., Bennett, B. M., et al. (1995) Electroporation of adherent cells in situ for the introduction of nonpermeant molecules. Methods Mol. Biol. 48, 93–113.
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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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308 Boyd et al. Fig. 3. Histogram o
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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
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Page 351 and 352: 332 Kobayashi 0.1% TFA at a flow ra
Page 353 and 354: 334 Kobayashi Both recombinant S-10
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Page 359 and 360: 340 Walsh et al. verts CaM from an
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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
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Page 385 and 386: 366 Persechini of the different CaM
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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
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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
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Page 425 and 426: 406 Török et al. the significantl
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412 Index Stern-Volmer plot, 78, 81
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414 Index Phenothiazines, see Calmo
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