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Fluorescence Ratio (520/490)x10000 Nedd8 and CUL1 reagents • Biotin-Nedd8 assay demonstrated low S/B and required end-point assay mode • FITC-Nedd8 is consistent with kinetic mode • Lumi4®-Tb anti-FLAG Ab has no effect on the reaction • CUL1 with N-terminal FLAG tag provides higher S/B in the assay than C-terminal FLAG tag 1500 1000 FLAG-SCF SCF-FLAG 500 0 0 50 100 150 200 [SCF] (nM)
R a tio (5 2 0 /4 9 0 )x 1 0 0 0 0 Fluorescence Ratio (520/490) x10000 S /B R a tio (5 2 0 /4 9 0 )x 1 0 0 0 0 S/B CUL1 neddylation assay development 6 0 0 0 6 6 0 0 0 6 4 0 0 0 4 4 0 0 0 4 1 5 m in 2 0 0 0 3 0 m in 6 0 m in 2 1 5 m in 3 0 m in 6 0 m in 2 0 0 0 1 5 m in 3 0 m in 5 0 m in 2 15 min 30 min 50 min 0 0 5 0 0 1 0 0 0 [flu o -N e d d 8 ] (n M ) 0 0 5 0 0 1 0 0 0 [flu o -N e d d 8 ] (n M ) 0 0 5 0 1 0 0 1 5 0 2 0 0 [S C F ] (n M ) 0 0 50 100 150 200 [SCF] (nM) 4000 9 0 0 0 L u m i4 -T b A b 0 .0 6 2 5 3000 0 nM NAE1 12.5 nM NAE1 50 nM NAE1 200 nM NAE1 6 0 0 0 0 .1 2 5 0 .2 5 2000 0 .5 3 0 0 0 1 1000 2 0 0 10 20 30 40 50 Time (min) • All assay parameters were optimized 0 0 1 0 2 0 3 0 4 0 5 0 [A T P ] (u M ) • Many reagents tested in “matrix” experiments
Page 1 and 2: HTRF Symposium Avignon, France Apri
Page 3 and 4: • SBMRI and CPCCG • NIH Roadmap
Page 5 and 6: Development of Sanford-Burnham Medi
Page 7 and 8: Sanford-Burnham’s Discovery Resea
Page 9 and 10: Conrad Prebys Center for Chemical G
Page 11 and 12: Sanford-Burnham uHTS Facility (Orla
Page 13 and 14: Specifics of MLPCN projects • MLP
Page 15 and 16: MLPCN screening at CPCCG Detection
Page 17 and 18: MLPCN UBL projects performed at CPC
Page 19 and 20: SUMO PPI project • PI: Yuan Chen,
Page 21 and 22: Assay design for SUMO PPI project B
Page 23 and 24: SUMO-PPI MLPCN screening project
Page 25 and 26: SUMOylation project: HTRF Assay GST
Page 27 and 28: SUMOylation project: Probe compound
Page 29: • Assay Reagents: Assay captures
Page 33 and 34: Z' factor CUL1 neddylation project
Page 35 and 36: MLN4924 has a complex mechanism of
Page 37 and 38: Concluding remarks • HTRF approac

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