Optimizing A Kinase Assay For Ikkbeta On An Hts Station (373 views)
J Biomol Screen (ISSN: 1087-0571, 1552-454x, 1552-454xelectronic), 2009 Dec; 14(10): 1263-1268.
Export to BibTeX Export to EndNote
Paper type: Journal Article, Research Support, Non-U. S. Gov'T,
Impact factor: 2.395, 5-year impact factor: 2.572
Url: http://www.scopus.com/inward/record.url?eid=2-s2.0-71949123743&partnerID=40&md5=1ca3dd845d14830ede439b2982d74d8b
Keywords: Hts, Ikkβ, Tr-Fret, Z′ Factor, Dimethyl Sulfoxide, Glutathione Transferase, Histidine, I Kappa B Kinase Beta, I Kappa B Kinase Inhibitor, Article, Controlled Study, Enzyme Activity, Enzyme Assay, Fluorescence Resonance Energy Transfer, High Throughput Screening, Priority Journal, High-Throughput Screening Assays, I-Kappa B Kinase, I-Kappa B Proteins, Time Factors, Z Factor, Methods, Metabolism,
Affiliations:
*** IBB - CNR ***
Istituto di Biostrutture e Bioimmagini, CNR, Via Mezzocannone, 16, 80134, Napoli, Italy
Dipartimento Delle Scienze Biologiche, Università di Napoli, Federico II, Napoli, Italy
References:
Strnad, J., Burke, J.R., IkappaB kinase inhibitors for treating autoimmune and inflammatory disorders: Potential and challenges (2007) Trends Pharmacol Sci, 28, pp. 142-14
Goldstein, D.M., Gray, N.S., Zarrinkar, P.P., High-throughput kinase profiling as a platform for drug discovery (2008) Nat Rev Drug Discov, 7, pp. 391-397
Oliver Von Ahsen, U.B., High-throughput screening for kinase inhibitors (2005) ChemBioChem, 6, pp. 481-490
Sera, T., Exploiting chemical libraries, structure, and genomics in the search for kinase inhibitors (2007) Tanpakushitsu Kakusan Koso, 52 (SUPPL.), pp. 1812-1813
Thaimattam, R., Banerjee, R., Miglani, R., Iqbal, J., Protein kinase inhibitors: Structural insights into selectivity (2007) Curr Pharm des, 13, pp. 2751-2765
Klumpp, M., Boettcher, A., Becker, D., Meder, G., Blank, J., Leder, L., Readout technologies for highly miniaturized kinase assays applicable to high-throughput screening in a 1536-well format (2006) J Biomol Screen, 11, pp. 617-633
Xu, Q.H., Wang, S., Korystov, D., Mikhailovsky, A., Bazan, G.C., Moses, D., The fluorescence resonance energy transfer (FRET) gate: A time-resolved study (2005) Proc Natl Acad Sci USA, 102, pp. 530-535
Moshinsky, D.J., Ruslim, L., Blake, R.A., Tang, F., Widely applicable, high-throughput TR-FRET assay for the measurement of kinase autophosphorylation: VEGFR-2 as a prototype (2003) J Biomol Screen, 8, pp. 447-452
Bazin, H., Trinquet, E., Mathis, G., Time resolved amplification of cryptate emission: A versatile technology to trace biomolecular interactions (2002) J Biotechnol, 82, pp. 233-250
Lance Ultra IKKβ Kinase Assay, , www.perkinelmer.com, Technical note U-TRF. Waltham, MA: PerkinElmer
Zhang, J.H., Chung, T.D., Oldenburg, K.R., A simple statistical parameter for use in evaluation and validation of high throughput screening assays (1999) J Biomol Screen, 4, pp. 67-73
Podolin, P.L., Callahan, J.F., Bolognese, B.J., Li, Y.H., Carlson, K., Davis, T.G., Attenuation of murine collagen-induced arthritis by a novel, potent, selective small molecule inhibitor of Iκb kinase 2, TPCA-1 (2-[(aminocarbonyl) amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide), occurs via reduction of proinflammatory cytokines and antigen-induced T cell proliferation (2005) J Pharmacol Exp Ther, 312, pp. 373-381
Goldstein, D. M., Gray, N. S., Zarrinkar, P. P., High-throughput kinase profiling as a platform for drug discovery (2008) Nat Rev Drug Discov, 7, pp. 391-397
Xu, Q. H., Wang, S., Korystov, D., Mikhailovsky, A., Bazan, G. C., Moses, D., The fluorescence resonance energy transfer (FRET) gate: A time-resolved study (2005) Proc Natl Acad Sci USA, 102, pp. 530-535
Moshinsky, D. J., Ruslim, L., Blake, R. A., Tang, F., Widely applicable, high-throughput TR-FRET assay for the measurement of kinase autophosphorylation: VEGFR-2 as a prototype (2003) J Biomol Screen, 8, pp. 447-452
Lance Ultra IKK Kinase Assay, , www. perkinelmer. com, Technical note U-TRF. Waltham, MA: PerkinElmer
Zhang, J. H., Chung, T. D., Oldenburg, K. R., A simple statistical parameter for use in evaluation and validation of high throughput screening assays (1999) J Biomol Screen, 4, pp. 67-73
Podolin, P. L., Callahan, J. F., Bolognese, B. J., Li, Y. H., Carlson, K., Davis, T. G., Attenuation of murine collagen-induced arthritis by a novel, potent, selective small molecule inhibitor of I b kinase 2, TPCA-1 (2- [(aminocarbonyl) amino] -5- (4-fluorophenyl) -3-thiophenecarboxamide), occurs via reduction of proinflammatory cytokines and antigen-induced T cell proliferation (2005) J Pharmacol Exp Ther, 312, pp. 373-381
J Biomol Screen (ISSN: 1087-0571, 1552-454x, 1552-454xelectronic), 2009 Dec; 14(10): 1263-1268.
Export to BibTeX Export to EndNote
Paper type: Journal Article, Research Support, Non-U. S. Gov'T,
Impact factor: 2.395, 5-year impact factor: 2.572
Url: http://www.scopus.com/inward/record.url?eid=2-s2.0-71949123743&partnerID=40&md5=1ca3dd845d14830ede439b2982d74d8b
Keywords: Hts, Ikkβ, Tr-Fret, Z′ Factor, Dimethyl Sulfoxide, Glutathione Transferase, Histidine, I Kappa B Kinase Beta, I Kappa B Kinase Inhibitor, Article, Controlled Study, Enzyme Activity, Enzyme Assay, Fluorescence Resonance Energy Transfer, High Throughput Screening, Priority Journal, High-Throughput Screening Assays, I-Kappa B Kinase, I-Kappa B Proteins, Time Factors, Z Factor, Methods, Metabolism,
Affiliations:
*** IBB - CNR ***
Istituto di Biostrutture e Bioimmagini, CNR, Via Mezzocannone, 16, 80134, Napoli, Italy
Dipartimento Delle Scienze Biologiche, Università di Napoli, Federico II, Napoli, Italy
References:
Strnad, J., Burke, J.R., IkappaB kinase inhibitors for treating autoimmune and inflammatory disorders: Potential and challenges (2007) Trends Pharmacol Sci, 28, pp. 142-14
Goldstein, D.M., Gray, N.S., Zarrinkar, P.P., High-throughput kinase profiling as a platform for drug discovery (2008) Nat Rev Drug Discov, 7, pp. 391-397
Oliver Von Ahsen, U.B., High-throughput screening for kinase inhibitors (2005) ChemBioChem, 6, pp. 481-490
Sera, T., Exploiting chemical libraries, structure, and genomics in the search for kinase inhibitors (2007) Tanpakushitsu Kakusan Koso, 52 (SUPPL.), pp. 1812-1813
Thaimattam, R., Banerjee, R., Miglani, R., Iqbal, J., Protein kinase inhibitors: Structural insights into selectivity (2007) Curr Pharm des, 13, pp. 2751-2765
Klumpp, M., Boettcher, A., Becker, D., Meder, G., Blank, J., Leder, L., Readout technologies for highly miniaturized kinase assays applicable to high-throughput screening in a 1536-well format (2006) J Biomol Screen, 11, pp. 617-633
Xu, Q.H., Wang, S., Korystov, D., Mikhailovsky, A., Bazan, G.C., Moses, D., The fluorescence resonance energy transfer (FRET) gate: A time-resolved study (2005) Proc Natl Acad Sci USA, 102, pp. 530-535
Moshinsky, D.J., Ruslim, L., Blake, R.A., Tang, F., Widely applicable, high-throughput TR-FRET assay for the measurement of kinase autophosphorylation: VEGFR-2 as a prototype (2003) J Biomol Screen, 8, pp. 447-452
Bazin, H., Trinquet, E., Mathis, G., Time resolved amplification of cryptate emission: A versatile technology to trace biomolecular interactions (2002) J Biotechnol, 82, pp. 233-250
Lance Ultra IKKβ Kinase Assay, , www.perkinelmer.com, Technical note U-TRF. Waltham, MA: PerkinElmer
Zhang, J.H., Chung, T.D., Oldenburg, K.R., A simple statistical parameter for use in evaluation and validation of high throughput screening assays (1999) J Biomol Screen, 4, pp. 67-73
Podolin, P.L., Callahan, J.F., Bolognese, B.J., Li, Y.H., Carlson, K., Davis, T.G., Attenuation of murine collagen-induced arthritis by a novel, potent, selective small molecule inhibitor of Iκb kinase 2, TPCA-1 (2-[(aminocarbonyl) amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide), occurs via reduction of proinflammatory cytokines and antigen-induced T cell proliferation (2005) J Pharmacol Exp Ther, 312, pp. 373-381
Goldstein, D. M., Gray, N. S., Zarrinkar, P. P., High-throughput kinase profiling as a platform for drug discovery (2008) Nat Rev Drug Discov, 7, pp. 391-397
Xu, Q. H., Wang, S., Korystov, D., Mikhailovsky, A., Bazan, G. C., Moses, D., The fluorescence resonance energy transfer (FRET) gate: A time-resolved study (2005) Proc Natl Acad Sci USA, 102, pp. 530-535
Moshinsky, D. J., Ruslim, L., Blake, R. A., Tang, F., Widely applicable, high-throughput TR-FRET assay for the measurement of kinase autophosphorylation: VEGFR-2 as a prototype (2003) J Biomol Screen, 8, pp. 447-452
Lance Ultra IKK Kinase Assay, , www. perkinelmer. com, Technical note U-TRF. Waltham, MA: PerkinElmer
Zhang, J. H., Chung, T. D., Oldenburg, K. R., A simple statistical parameter for use in evaluation and validation of high throughput screening assays (1999) J Biomol Screen, 4, pp. 67-73
Podolin, P. L., Callahan, J. F., Bolognese, B. J., Li, Y. H., Carlson, K., Davis, T. G., Attenuation of murine collagen-induced arthritis by a novel, potent, selective small molecule inhibitor of I b kinase 2, TPCA-1 (2- [(aminocarbonyl) amino] -5- (4-fluorophenyl) -3-thiophenecarboxamide), occurs via reduction of proinflammatory cytokines and antigen-induced T cell proliferation (2005) J Pharmacol Exp Ther, 312, pp. 373-381
Optimizing A Kinase Assay For Ikkbeta On An Hts Station
Using a commercially available time-resolved fluorescence resonance energy transfer (TR-FRET)-based assay for IKKβ, the authors have automated the assay procedure on a high-throughput screening station to carry out screening campaigns on multiwell plates. They have determined the Z′ factor and optimized volumes, times, and time-resolved fluorescence parameters. They have also compared 2 kinases with different fusion tags, the influence of different enzyme/substrate ratios and of DMSO presence at different concentration. The authors found that glutathione S-transferase (GST)-fused IKKβ shows better signal-to-noise (S/N) ratios over the poly-histidine-tagged variant. The substrate can be used at 50 nM with optimal performances when the enzyme is used at 2 nM. DMSO at 0.2% and 1% only slightly affects the S/N ratio, whereas when used at 2%, the final concentration deriving from a 50-fold dilution from a 5-mM stock solution in pure solvent, S/N undergoes a decrease of about 15%. Under the optimized conditions, the assay Z′ factor calculated over 192 data points has an optimized value of 0.881 and allows the testing of 94 molecules in quadruplicate in 140 min. © 2009 Society for Biomolecular Sciences.
Using a commercially available time-resolved fluorescence resonance energy transfer (TR-FRET)-based assay for IKKβ, the authors have automated the assay procedure on a high-throughput screening station to carry out screening campaigns on multiwell plates. They have determined the Z′ factor and optimized volumes, times, and time-resolved fluorescence parameters. They have also compared 2 kinases with different fusion tags, the influence of different enzyme/substrate ratios and of DMSO presence at different concentration. The authors found that glutathione S-transferase (GST)-fused IKKβ shows better signal-to-noise (S/N) ratios over the poly-histidine-tagged variant. The substrate can be used at 50 nM with optimal performances when the enzyme is used at 2 nM. DMSO at 0.2% and 1% only slightly affects the S/N ratio, whereas when used at 2%, the final concentration deriving from a 50-fold dilution from a 5-mM stock solution in pure solvent, S/N undergoes a decrease of about 15%. Under the optimized conditions, the assay Z′ factor calculated over 192 data points has an optimized value of 0.881 and allows the testing of 94 molecules in quadruplicate in 140 min. © 2009 Society for Biomolecular Sciences.
Optimizing A Kinase Assay For Ikkbeta On An Hts Station
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Optimizing A Kinase Assay For Ikkbeta On An Hts Station
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9 Records (9 excluding Abstracts).
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Total impact factor: 37.5 (37.5 excluding Abstracts).
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