Stephanie Bechtel German Cancer Research Center Molecular Genome Analysis
Im Neuenheimer Feld 580
Heidelberg Germany s.bechtel@dkfz-heidelberg.de Genome-wide p38 RNAi screen activation or inhibition of P38 MAPK signaling by knock-down of endogenously expressed genes/proteins - whole genome luciferase reporter gene assay reflecting the activation state of the P38 MAPK signaling Not defined A Genome-wide RNAi screen to identify modulators of human P38 signalling using a human siRNA library containing 21.000 different siRNA pools Tissue culture flask with "peel-off" lids 15000 1 TPP 90551 CulturePlate-96 96 PerkinElmer 6005680 PS-Microplate 96 32 96 Greiner 655161 HeLa ATCC CCL-2
15 false DMEM/HIGH GIBCO 31053 143992 FBS (Fetal bovine Serum) GIBCO 16000-044 1404590 L-Glutamine Invitrogen 25030-024 0.2 Penicillin/Streptomycin Invitrogen/GIBCO 15070-063 1280788 Hygromycin Invitrogen/GIBCO 10687-010 A8906 50 G418 GIBCO 14040 1127731 50 OptiMEM Invitrogen 31985-047 HyperFectTransfectionReagent QIAGEN 301707 127150835 GeneXYZ siRNA Dharmacon @@@ 2x10e-6 TNF alpha Sigma T0157 0.01 Tricine Sigma 0.5 ATP AppliChem A1348,0025 0.1 MgSO4 Sigma 0.1 EDTA Sigma 0.01 DTT Roche 708984 0.2 Coenzyme A AppliChem A0813,0100 0.02 Luciferin AppliChem A1006,0100 0.025 10x PBS GIBCO SH30378.02 AQM25314 M-PER Mammalian Protein Extraction Reagent Perbio 78501 Micro BCA Protein Assay Kit Perbio 23235 Growth Medium r1 r2 r3 r4 r5 r6 flask Assay Medium transfer in 96well format r7 r2 r3 siRNA/TransfectionComplex perturbation r8 r9 r7 Starvation Medium treatment or perturbation r7 r2 r3 TNF alpha stimulation treatment or perturbation r7 r2 r3 r10 Luciferase Assay Reagent r11 r12 r13 r14 r15 r16 r17 Fusion A153603 plate reader Perkin Elmer 427339 SpectraMax 190 plate reader Molecular Devices 02364 cell propagation as1 cr1 cl1 37 5 95 48 siRNA transfection as2 cr2 cr3 7500 37 5 95 48 starvation as2 cr4 37 5 95 24 TNF alpha stimulation as2 cr5 37 5 95 3 PBSwash as2 r18 21 0.02 cell lysis as2 r19 21 0.06 Luciferase assay as2 cr6 21 0.02 quantification of total protein concentration as3 r20 37 2 i1 CulturPlate-96 luminescence by plate i2 PS-Microplate 96