Target Discovery and Validation Reviews and Protocols

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Transfected Cell Arrays 157 Fig. 1. Five basal steps of the method: (1) Preparation of the transfection solutions on an automated liquid handler with a 96-pipet head. (2) Spotting of the transfection solutions with a spotting Robot, e.g. ChipWriter Compact, on Lab-Tek chambered coverglass. (3) Plating of the cells on dishes with the dried transfection solutions. (4) Preparation of samples (manually or automatically) for functional analysis, e.g., immunostaining. (5) Analysis of samples by high-content screening microscopy.
158 Erfle and Pepperkok cells away from the spot region. To retrieve the spot regions and to highlight successfully transfected cells of siRNA experiments, a Cy3-labeled DNA oligonucleotide is used as cotransfection marker. In this case, 0.5 µL of a 40 µM marker solution is included in step 1 of the protocol, resulting in a total oligonucleotide volume of 1.5 µL (1 µL of siRNA plus 0.5 µL of Cy3-labeled oligonucleotide). With this protocol, it is possible to cotransfect plasmid cDNA and siRNA. In this case, 1 µL of siRNA plus 1 µL of plasmid cDNA are added in step 1, resulting in a total volume of 2 µL. 1. Add 1 µL of the respective siRNA stock solution (20 µM in RNA dilution buffer as supplied by the manufacturer) to each well. For plasmid transfections, 1 µL of plasmid DNA at a stock concentration of 500 ng/µL is added. 2. Add 7.5 µL EC buffer (part of the Effectene transfection kit, QIAGEN) containing 0.2 M sucrose and mix thoroughly by pipeting three times up and down. The EC buffer can be replaced by water, also containing 0.2 M sucrose for LipofectAMINE 2000 (Invitrogen). 3. Incubate the mixture for 10 min at room temperature. 4. Add 4.5 µL of Effectene transfection reagent (QIAGEN) or 3.5 µL of LipofectAMINE 2000 (Invitrogen). 5. Incubate for 10 min at room temperature. 6. Add 7.25 µL of 0.08% gelatin (G-9391, Sigma) containing 3.5 × 10 –4 % fibronectin (Sigma). 7. Spin down the 384-well plate for 1 min at 216g. The solution is now ready for the spotting process. 3.2. Spotting the Transfection Solution on Uncoated Lab-Tek Dishes As a substrate, we use untreated chambered coverglass dishes, allowing live cell imaging and easy-to-perform immunostaining. We use a ChipWriter Compact robot (Bio-Rad, Hercules, CA) equipped with PTS 600 (Point Technologies) solid pins. The distance between the spots is 1125 µm, resulting in spot diameters of approx 450 µm. The spotted solutions on the Lab-Tek chamber are dried at room temperature for at least 12 h after printing before cells are plated. The solutions of one 384-well plate (see Subheading 3.1.) are sufficient to spot at least 50 identical Lab-Tek chambers, which can be stored for later use in a dry environment for several months without obvious loss in transfection efficiencies. The outer dimensions of a Lab-Tek chamber are 57.2 × 25.6 mm (length × width). Of this area we use, 34.875 mm in length and 12.375 mm in width to spot 384 samples. Spotting 384 samples on 48 Lab-Tek chambers, by using eight solid pins in the x-direction mode typically lasts 6 h. 1. We adjust the temperature of the 384-well plate with an in-house–built watercooled plate to 12°C.
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METHODS IN MOLECULAR BIOLOGY 360 T
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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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© 2007 Humana Press Inc. 999 River
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vi Preface get. Approaches to targe
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viii Contents 10 Transgenic Animal
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x Contributors SAHOHIME MATSUMOTO
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xii Contents of Volume 2 12 Validat
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2 Sioud disease pathogenesis and pe
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4 Sioud A more fruitful approach fo
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6 Sioud both transient and permanen
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8 Sioud serum biomarkers. This syne
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10 Sioud Fig. 2. Schematic of targe
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12 Sioud 13. Magin, T. M. (1998) Le
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Harnessing the Human Genome 15 The
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Harnessing the Human Genome 17 Fig.
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Harnessing the Human Genome 23 The
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Harnessing the Human Genome 27 repr
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Harnessing the Human Genome 29 14.
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Harnessing the Human Genome 31 45.
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34 Imoto et al. (4,5), and Bayesian
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36 Imoto et al. 2. Materials Two ty
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38 Imoto et al. Fig. 3. Graphical v
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40 Imoto et al. Fig. 5. Result of t
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42 Imoto et al. p(D |G) = ∫ p(D,
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44 Imoto et al. β k (k = 1,…,q j
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46 Imoto et al. Fig. 7. Partial res
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48 Imoto et al. Fig. 8. Strategy to
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50 Imoto et al. Fig. 9. (continued)
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52 Imoto et al. Table 3 List of Roo
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54 Imoto et al. 5. De Hoon, M. J. L
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56 Imoto et al. 39. Kamimura, T., S
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58 Beaty et al. cytotoxic drugs and
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60 Beaty et al. Fig. 1. Principles
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62 Beaty et al. oligonucleotide mic
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64 Beaty et al. 3. The 12% polyacry
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66 Beaty et al. 2.3.3. Protein Stai
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68 Beaty et al. 3.1.3. Labeling of
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70 Beaty et al. 1 µL of the anneal
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72 Beaty et al. 4. Phenol:cholorfor
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74 Beaty et al. 19. Wash twice with
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76 Beaty et al. The Following Volum
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78 Beaty et al. using a protein ass
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80 Beaty et al. 6. Shake the gel fo
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82 Beaty et al. Fig. 2. Preparation
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84 Beaty et al. search in. A widely
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86 Beaty et al. 3. Kern, S. E., Hru
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88 Beaty et al. 34. Peters, D. G.,
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5 Molecular Classification of Breas
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Molecular Profiling of Breast Cance
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Fig. 2. (Continued) the right ident
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Page 120 and 121: Molecular Profiling of Breast Cance
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Page 168 and 169: 9 Production of siRNA- and cDNA-Tra
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Page 191 and 192: 178 Houdebine systems. Moreover, st
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Page 217 and 218: 204 Vijayaraj, Söhl, and Magin 1.
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208 Vijayaraj, Söhl, and Magin fil
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210 Table 1 Orthologous Human and M
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212 Table 2 Orthologous Human and M
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214 Vijayaraj, Söhl, and Magin ulc
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216 Vijayaraj, Söhl, and Magin of
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218 Vijayaraj, Söhl, and Magin sug
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220 Vijayaraj, Söhl, and Magin abs
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222 Vijayaraj, Söhl, and Magin 1.2
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224 Vijayaraj, Söhl, and Magin Fig
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226 Vijayaraj, Söhl, and Magin gen
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228 Vijayaraj, Söhl, and Magin the
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230 Vijayaraj, Söhl, and Magin f.
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232 Vijayaraj, Söhl, and Magin 3.
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234 Vijayaraj, Söhl, and Magin b.
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240 Vijayaraj, Söhl, and Magin alt
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242 Table 3 Time Schedule For Aggre
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250 Vijayaraj, Söhl, and Magin 101
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12 The HUVEC/Matrigel Assay An In V
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256 Skovseth, Küchler, and Haralds
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270 Iversen and Sørensen witnessed
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272 Iversen and Sørensen Fig. 1. P
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274 Iversen and Sørensen the core
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14 An Overview of the Immune System
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Overview of Immune System 279 diffe
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Overview of Immune System 281 Fig.
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Overview of Immune System 283 then
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Overview of Immune System 285 expre
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Overview of Immune System 287 Once
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Overview of Immune System 289 solub
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Overview of Immune System 291 amino
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Overview of Immune System 293 (unre
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Overview of Immune System 297 the c
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Overview of Immune System 299 (44).
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Overview of Immune System 301 demon
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Overview of Immune System 307 some
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Overview of Immune System 309 sera
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Overview of Immune System 313 measu
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Overview of Immune System 315 42. H
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Overview of Immune System 317 74. D
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15 Potential Target Antigens for Im
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Tumor Antigen Discovery 321 develop
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Tumor Antigen Discovery 323 panel b
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Tumor Antigen Discovery 325 16. Joc
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16 Identification of Tumor Antigens
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Immunoproteomics 329 by “shot gun
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Immunoproteomics 331 isoelectric fo
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Immunoproteomics 333 3. 2D-PAGE is
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17 Protein Arrays A Versatile Toolb
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Protein Arrays 337 Table 1 Classifi
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Protein Arrays 339 fractions from c
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Protein Arrays 341 combinatorial sc
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Protein Arrays 343 The ideal proteo
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Protein Arrays 345 18. Cekaite, H.
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Protein Arrays 347 49. Yuko Kawahas
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Index 349 Index A Acetyl-CoA carbox
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Index 351 conditional by using FRT
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Index 353 Recombinant tumor antigen
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