Target Discovery and Validation Reviews and Protocols

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Transfected Cell Arrays 159 2. The dwell time the spotter pins remain in the source plate is set to 0.5 s. 3. The dwell time the spotter pins remain on the Lab-Tek chamber is set to 0.1 s. 4. The washing procedures between the spotting of distinct samples are set as follows: a. Wash container: 10 s. b. Sonication container: 10 s. c. Vacuum drying: 10 s. 3.3. Plating of Cells on Lab-Tek Dishes With Dried Transfection Solution The density of the cells plated on the dried spots of a Lab-Tek chamber is always a compromise between the improved statistics that can be achieved with high cell densities and the quality of the microscopic analyses that is best at low cell densities. 1. Typically, we plate 1.25 × 10 5 actively growing HeLa, MCF7, COS7L, or HEK293 cells in 2.5 mL of culture medium (Dulbecco’s modified Eagle’s medium containing 10% heat-inactivated fetal calf serum, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/mL streptomycin) on the dried spots of one Lab-Tek culture dish. This process results in approx 100 to 200 cells residing on one spot. 2. The incubation time (at 37°C and 5% CO 2 ) for the successful expression of plasmid cDNAs varies between 12 and 24 h. 3. The incubation time for RNA interference (RNAi) experiments varies between 20 to 50 h and strongly depends on the stability of the proteins targeted by the siRNAs spotted. For long-term experiments lasting several days, e.g., RNAi experiments targeting stable proteins, the cell density needs to be lowered compared with the density typically used (see step 1), because cells may stop growing because of contact inhibition at later time-points of the experiment (e.g., 60 h), which makes experiments addressing cell cycle- or signal transduction-related questions difficult to interpret. 3.4. Preparation of Samples for Functional Analysis For functional analysis involving high-content screening microscopy, we frequently use immunofluorescence (6) to monitor molecule-specific morphological and biochemical parameters. The immunostaining procedure in Lab-Tek chambered glass cover slips is very effective and cheap because it can be performed with the same antibody for hundreds of target genes in parallel. 1. For the staining of Lab-Tek chambers, we apply 250 µL of the correspondingantibody by carefully distributing the fluid over the spotted area. 2. Incubate for 10 min with the lid closed to avoid rapid evaporation. 3. Wash two times with 2 mL of phosphate-buffered saline (PBS) (30 min each). 4. Stain cell nuclei with 0.2 µg/mL Hoechst B2261 (Sigma). The strong nuclear staining achieved in this way facilitates automated focusing during image acquisition (3) (see Subheading 3.5.).
160 Erfle and Pepperkok 5. The stained samples are stored at 4°C either embedded in Mowiol or in PBS solution containing 0.1% azide after a brief poststaining fixation of the samples with paraformaldehyde for 2 min. 3.5. Analysis of Samples by High-Content Screening In principle, images of the cells on the spots can be acquired with any commercially available inverted microscope. We use a ScanR (Olympus Biosystems, Munich, Germany; [3]) scanning microscope, with automated focus, allowing time-lapse data acquisition. The microscope is equipped with standard filters to detect DAPI, GFP, and Cy3. A 10 × /0.4 air or a 40 × /0.95 air PlanApo objective (Olympus) is used for image acquisition. A key step of the whole image acquisition process is to find the first spot of the array. We use the fluorescent signal of the cotransfected Cy3 DNA oligonucleotide. In addition, we mark the first spot manually with a thin and waterresistant black marker pen on the opposite side of the coverglass where the spots are located, before cell seeding. 1. Assign the spot-to-spot distance, the number of samples, and the array dimensions (number of samples in x-direction and number of samples in y-direction). 2. For image processing the images are background corrected by subtracting the average pixel value in a blank region of the image. The locations of the cells are determined by Hoechst nuclear stain. A possible fluorescence statistics can be based on the mean fluorescence values in a dilated region around the nucleus (including or not including nucleus, depending on the assay). Average values obtained should be normalized to the siRNA control (1.0) (nonsilencing) spotted on the same slide. 4. Notes 1. The spotting robot used has to be able to pass the walls of the Lab-Tek chamber. 2. To achieve the required accuracy of the spot-to-spot distance, high-resolution spotting robots are necessary. References 1. Ziauddin, J. and Sabatini, D. M. (2001) Microarrays of cells expressing defined cDNAs. Nature 411, 107–110. 2. Erfle, H., Simpson, J. C., Bastiaens, P. I., and Pepperkok, R. (2004) siRNA cell arrays for high-content screening microscopy. BioTechniques 37, 454–462. 3. Liebel, U., Starkuviene, V., Erfle, H., et al. (2003) A microscope-based screening platform for large scale functional analysis in intact cells. FEBS Lett. 554, 394–398. 4. Starkuviene, V., Liebel, U., Simpson, J. C., et al. (2004) High-content screening microscopy identifies novel proteins with a putative role in secretory membrane traffic. Genome Res. 14, 1948–1956.
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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 122 and 123: Molecular Profiling of Breast Cance
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Page 217 and 218: 204 Vijayaraj, Söhl, and Magin 1.
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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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222 Vijayaraj, Söhl, and Magin 1.2
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230 Vijayaraj, Söhl, and Magin f.
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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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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 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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