Protein Engineering Protocols - Mycobacteriology research center

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<strong>Protein</strong>-Based Ca 2+ Indicators 77Fig. 3. Example fluorescence spectrum of YC6.1.Fig. 4. Example Ca 2+ -binding curve of YC6.1.3. To produce the Ca 2+ -binding curve, plot the free[Ca 2+ ] vs the change in the emissionratio. Figure 4 shows the Ca 2+ -binding curve of YC6.1.3.3. Ca 2+ Imaging With ChameleonsThe following section describes a simple Ca 2+ -imaging experiment usingHeLa cells; however, with minor modifications, the method can be applied to
78 Truong et al.Fig. 5. Digital fluorescence microscope facilities.other adherent cells and physiological contexts. In this section, we describe thepreparation of the cell culture (see Subheading 3.3.1.) and the acquisition ofdata (see Subheading 3.3.2.).3.3.1. Preparation of the Cell Culture1. Plate HeLa cells on 35-mm-diameter glass-bottom dishes with DMEM–10% FBSmedia.2. Incubate the cells at 37°C (5% CO 2) until cells are 50 to 80% confluent.3. Transfect cells with the mammalian expression plasmid containing your chameleon,using GeneJuice (Novagen).4. Remove the transfection mixture after 6 h and replace with 1.5 mL of DMEM–10%FBS media.5. Incubate the cells at 37°C (5% CO 2) for 24 h. The cells are ready to perform theCa 2+ -imaging experiment.3.3.2. Data AcquisitionThe experiment should be performed in a dark room to reduce backgroundlight. Figure 5 shows an image of our microscopy facilities.1. Turn on the Xenon lamp, microscope, filter changers, and computer.2. Remove the growth media from the culture dish.3. Wash with 1 mL of HBSS (+CaCl 2).4. Add 1 mL of fresh HBSS (+CaCl 2).
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METHODS IN MOLECULAR BIOLOGY 352Pr
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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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PrefaceProtein engineering is a fas
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ContentsPreface ...................
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ContributorsKATJA M. ARNDT • Inst
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Contributors xiKAZUNARI TAIRA • D
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1Combinatorial Protein Design Strat
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Combinatorial Protein Design Strate
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2Global Incorporation of Unnatural
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Incorporation of Unnatural Amino Ac
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Page 48 and 49: 3Considerations in the Design and O
Page 50 and 51: Design of Coiled Coil Structures 37
Page 84 and 85: 4Calcium Indicators Based on Calmod
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Page 94 and 95: Protein-Based Ca 2+ Indicators 8145
Page 96 and 97: 5Design and Synthesis of Artificial
Page 98 and 99: Design of Zinc Finger Proteins 853.
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Page 109 and 110: 96 Koide and Koidewhile retaining t
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Page 113 and 114: 100 Koide and Koidetarget-binding s
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Page 117 and 118: 104 Koide and Koideup to 1 mM for h
Page 119 and 120: 106 Koide and Koideplate. Incubate
Page 121 and 122: 108 Koide and Koide1. Perform steps
Page 124 and 125: 7Engineering Site-Specific Endonucl
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8Protein Library Design and Screeni
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Protein Library Design and Screenin
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135Fig. 2. Excel worksheet describi
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137Fig. 4. Excel worksheet describi
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9Protein Design by Binary Patternin
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Protein Design by Binary Patterning
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10Versatile DNA Fragmentation and D
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NExT DNA Shuffling 169This chapter
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NExT DNA Shuffling 1713. Methods3.1
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NExT DNA Shuffling 17372°C, 4 min.
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NExT DNA Shuffling 175Fig. 2. Varia
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NExT DNA Shuffling 1773.5.1. Direct
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NExT DNA Shuffling 179Fig. 3. Reass
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181
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NExT DNA Shuffling 183likelihood of
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NExT DNA Shuffling 1853.9.2. Calibr
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NExT DNA Shuffling 187staining. Bec
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NExT DNA Shuffling 1894. Zhao, H.,
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11Degenerate Oligonucleotide Gene S
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Degenerate Oligonucleotide Gene Shu
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12M13 Bacteriophage Coat Proteins E
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Engineered M13 Bacteriophage Coat P
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222 Fujita et al.The methods that h
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224 Fujita et al.3. Streptavidin an
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226 Fujita et al.Fig. 2. (Continued
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228 Fujita et al.Fig. 3. (Continued
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230 Fujita et al.Fig. 3. (A) Schema
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232 Fujita et al.1. Add 2 µg mRNA
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234 Fujita et al.Innovative Bioscie
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236 Fujita et al.30. Kim, Y., Mlsa,
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238 Ghadessy and Holligeraffinity m
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240 Ghadessy and HolligerFig. 1. (A
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242 Ghadessy and HolligerFinally, t
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244 Ghadessy and Holliger2. Prepare
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246 Ghadessy and Holliger2. After 6
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248 Ghadessy and Holliger21. Oberho
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250 Campbell-Valois and Michnickscr
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252 Campbell-Valois and Michnickfol
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254 Campbell-Valois and Michnick7.
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256 Campbell-Valois and MichnickFig
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258 Campbell-Valois and Michnickres
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260Fig. 3. BL21 pREP4 cells were co
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262 Campbell-Valois and MichnickFig
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264 Campbell-Valois and Michnickvar
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276 Hecky et al.improved half-life
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278 Hecky et al.Fig. 1. (A) Scheme
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280 Hecky et al.11. Reverse primer
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282 Hecky et al.high variability in
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284 Hecky et al.coding sequence for
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286 Hecky et al.4. Analyze the resu
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Table 1Amino Acid Substitutions Sel
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290 Hecky et al.Fig. 4. Structure o
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292 Hecky et al.Fig. 5. Expression
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294 Hecky et al.Table 2Kinetic Para
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296 Hecky et al.The thermoactivity
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298 Hecky et al.Fig. 8. Unfolding o
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300 Hecky et al.for the first trans
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302 Hecky et al.7. Thompson, M. J.
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304 Hecky et al.40. Wang, X., Minas
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306 IndexCCalcium indicators, see C
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308 Indexchemiocompetent cellprepar
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310 Indexmaterials, 169, 170mutatio
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312 IndexTerminal truncation, see a
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