Nonradioactive Assay of FLAG®-Tagged MAPK Using ANTI-FLAG® Antibody-Coated Multiwell Plates

We have developed a rapid, sensitive, and quantitative 96-well microplate-based nonradioactive immunoprecipitation/kinase assay to evaluate mitogen-activated protein kinase (MAPK) activity. Three quantitative nonradioactive imunoprecipitation/kinase assays of MAPK were demonstrated on a 96-well micr...
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Gespeichert in:

Autor*in:	L. Zhang [verfasserIn] S. Uder [verfasserIn] T. Juehne [verfasserIn] B. Brizzard [verfasserIn] K. Song [verfasserIn]

Format:	E-Artikel
Sprache:	Englisch

Erschienen:	2002

Übergeordnetes Werk:	In: BioTechniques - Future Science Ltd, 2019, 32(2002), 2, Seite 442-447
Übergeordnetes Werk:	volume:32 ; year:2002 ; number:2 ; pages:442-447

Links:	Link aufrufen Link aufrufen Link aufrufen Journal toc Journal toc

DOI / URN:	10.2144/02322pf02

Katalog-ID:	DOAJ016194012

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spelling	10.2144/02322pf02 doi (DE-627)DOAJ016194012 (DE-599)DOAJ7e10e6cd099c46f4bb32c8ba528a9bd3 DE-627 ger DE-627 rakwb eng QH301-705.5 L. Zhang verfasserin aut Nonradioactive Assay of FLAG®-Tagged MAPK Using ANTI-FLAG® Antibody-Coated Multiwell Plates 2002 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier We have developed a rapid, sensitive, and quantitative 96-well microplate-based nonradioactive immunoprecipitation/kinase assay to evaluate mitogen-activated protein kinase (MAPK) activity. Three quantitative nonradioactive imunoprecipitation/kinase assays of MAPK were demonstrated on a 96-well microplate coated with ANTI-FLAG® M2 antibody (ANTI-FLAG M2 plate): (i) the capture of phosphorylated FLAG®-tagged MAPK fusion protein (FLAG-MAPK) from phorbol esters-stimulated, FLAG-MAPK-transfected COS-7 cells, coupled with a very sensitive ELISA procedure to quantitate the level of phosphorylation of FLAG-MAPK; (ii) the in vitro kinase reaction of FLAG-MAPK activity with a substrate and ATP in the same well used to captured the phosphorylated FLAG-MAPK; and (iii) the in vitro kinase reaction of captured non-activated FLAG-MAPK by its upstream kinase from phorbol 12-myristate 13-acetate (PMA)-stimulated COS-7 cells. These results demonstrate that the ANTI-FLAG M2 plate allows for the rapid and quantitative determination of phosphorylation of FLAG-MAPK directly from stimulated, transfected cell lysate. Captured, phosphorylated FLAG-MAPK retains catalytic activity as demonstrated by the phosphorylation of Elk-1 in the same well. Furthermore, phosphorylation of captured FLAG-MAPK by the upstream kinases can be observed directly on the plate. These assays are sensitive, specific, and suitable for handling multiple samples. Thus, the ANTI-FLAG M2 plate forms the basis of a high-throughput screening platform in kinase analysis. Biology (General) S. Uder verfasserin aut T. Juehne verfasserin aut B. Brizzard verfasserin aut K. Song verfasserin aut In BioTechniques Future Science Ltd, 2019 32(2002), 2, Seite 442-447 (DE-627)306320746 (DE-600)1496354-1 19409818 nnns volume:32 year:2002 number:2 pages:442-447 https://doi.org/10.2144/02322pf02 kostenfrei https://doaj.org/article/7e10e6cd099c46f4bb32c8ba528a9bd3 kostenfrei https://www.future-science.com/doi/10.2144/02322pf02 kostenfrei https://doaj.org/toc/0736-6205 Journal toc kostenfrei https://doaj.org/toc/1940-9818 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_370 GBV_ILN_602 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 32 2002 2 442-447
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allfieldsSound	10.2144/02322pf02 doi (DE-627)DOAJ016194012 (DE-599)DOAJ7e10e6cd099c46f4bb32c8ba528a9bd3 DE-627 ger DE-627 rakwb eng QH301-705.5 L. Zhang verfasserin aut Nonradioactive Assay of FLAG®-Tagged MAPK Using ANTI-FLAG® Antibody-Coated Multiwell Plates 2002 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier We have developed a rapid, sensitive, and quantitative 96-well microplate-based nonradioactive immunoprecipitation/kinase assay to evaluate mitogen-activated protein kinase (MAPK) activity. Three quantitative nonradioactive imunoprecipitation/kinase assays of MAPK were demonstrated on a 96-well microplate coated with ANTI-FLAG® M2 antibody (ANTI-FLAG M2 plate): (i) the capture of phosphorylated FLAG®-tagged MAPK fusion protein (FLAG-MAPK) from phorbol esters-stimulated, FLAG-MAPK-transfected COS-7 cells, coupled with a very sensitive ELISA procedure to quantitate the level of phosphorylation of FLAG-MAPK; (ii) the in vitro kinase reaction of FLAG-MAPK activity with a substrate and ATP in the same well used to captured the phosphorylated FLAG-MAPK; and (iii) the in vitro kinase reaction of captured non-activated FLAG-MAPK by its upstream kinase from phorbol 12-myristate 13-acetate (PMA)-stimulated COS-7 cells. These results demonstrate that the ANTI-FLAG M2 plate allows for the rapid and quantitative determination of phosphorylation of FLAG-MAPK directly from stimulated, transfected cell lysate. Captured, phosphorylated FLAG-MAPK retains catalytic activity as demonstrated by the phosphorylation of Elk-1 in the same well. Furthermore, phosphorylation of captured FLAG-MAPK by the upstream kinases can be observed directly on the plate. These assays are sensitive, specific, and suitable for handling multiple samples. Thus, the ANTI-FLAG M2 plate forms the basis of a high-throughput screening platform in kinase analysis. Biology (General) S. Uder verfasserin aut T. Juehne verfasserin aut B. Brizzard verfasserin aut K. Song verfasserin aut In BioTechniques Future Science Ltd, 2019 32(2002), 2, Seite 442-447 (DE-627)306320746 (DE-600)1496354-1 19409818 nnns volume:32 year:2002 number:2 pages:442-447 https://doi.org/10.2144/02322pf02 kostenfrei https://doaj.org/article/7e10e6cd099c46f4bb32c8ba528a9bd3 kostenfrei https://www.future-science.com/doi/10.2144/02322pf02 kostenfrei https://doaj.org/toc/0736-6205 Journal toc kostenfrei https://doaj.org/toc/1940-9818 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_370 GBV_ILN_602 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 32 2002 2 442-447
language	English
source	In BioTechniques 32(2002), 2, Seite 442-447 volume:32 year:2002 number:2 pages:442-447
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callnumber-first	Q - Science
author	L. Zhang
spellingShingle	L. Zhang misc QH301-705.5 misc Biology (General) Nonradioactive Assay of FLAG®-Tagged MAPK Using ANTI-FLAG® Antibody-Coated Multiwell Plates
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topic_title	QH301-705.5 Nonradioactive Assay of FLAG®-Tagged MAPK Using ANTI-FLAG® Antibody-Coated Multiwell Plates
topic	misc QH301-705.5 misc Biology (General)
topic_unstemmed	misc QH301-705.5 misc Biology (General)
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title	Nonradioactive Assay of FLAG®-Tagged MAPK Using ANTI-FLAG® Antibody-Coated Multiwell Plates
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title_full	Nonradioactive Assay of FLAG®-Tagged MAPK Using ANTI-FLAG® Antibody-Coated Multiwell Plates
author_sort	L. Zhang
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author_browse	L. Zhang S. Uder T. Juehne B. Brizzard K. Song
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doi_str_mv	10.2144/02322pf02
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title_sort	nonradioactive assay of flag®-tagged mapk using anti-flag® antibody-coated multiwell plates
callnumber	QH301-705.5
title_auth	Nonradioactive Assay of FLAG®-Tagged MAPK Using ANTI-FLAG® Antibody-Coated Multiwell Plates
abstract	We have developed a rapid, sensitive, and quantitative 96-well microplate-based nonradioactive immunoprecipitation/kinase assay to evaluate mitogen-activated protein kinase (MAPK) activity. Three quantitative nonradioactive imunoprecipitation/kinase assays of MAPK were demonstrated on a 96-well microplate coated with ANTI-FLAG® M2 antibody (ANTI-FLAG M2 plate): (i) the capture of phosphorylated FLAG®-tagged MAPK fusion protein (FLAG-MAPK) from phorbol esters-stimulated, FLAG-MAPK-transfected COS-7 cells, coupled with a very sensitive ELISA procedure to quantitate the level of phosphorylation of FLAG-MAPK; (ii) the in vitro kinase reaction of FLAG-MAPK activity with a substrate and ATP in the same well used to captured the phosphorylated FLAG-MAPK; and (iii) the in vitro kinase reaction of captured non-activated FLAG-MAPK by its upstream kinase from phorbol 12-myristate 13-acetate (PMA)-stimulated COS-7 cells. These results demonstrate that the ANTI-FLAG M2 plate allows for the rapid and quantitative determination of phosphorylation of FLAG-MAPK directly from stimulated, transfected cell lysate. Captured, phosphorylated FLAG-MAPK retains catalytic activity as demonstrated by the phosphorylation of Elk-1 in the same well. Furthermore, phosphorylation of captured FLAG-MAPK by the upstream kinases can be observed directly on the plate. These assays are sensitive, specific, and suitable for handling multiple samples. Thus, the ANTI-FLAG M2 plate forms the basis of a high-throughput screening platform in kinase analysis.
abstractGer	We have developed a rapid, sensitive, and quantitative 96-well microplate-based nonradioactive immunoprecipitation/kinase assay to evaluate mitogen-activated protein kinase (MAPK) activity. Three quantitative nonradioactive imunoprecipitation/kinase assays of MAPK were demonstrated on a 96-well microplate coated with ANTI-FLAG® M2 antibody (ANTI-FLAG M2 plate): (i) the capture of phosphorylated FLAG®-tagged MAPK fusion protein (FLAG-MAPK) from phorbol esters-stimulated, FLAG-MAPK-transfected COS-7 cells, coupled with a very sensitive ELISA procedure to quantitate the level of phosphorylation of FLAG-MAPK; (ii) the in vitro kinase reaction of FLAG-MAPK activity with a substrate and ATP in the same well used to captured the phosphorylated FLAG-MAPK; and (iii) the in vitro kinase reaction of captured non-activated FLAG-MAPK by its upstream kinase from phorbol 12-myristate 13-acetate (PMA)-stimulated COS-7 cells. These results demonstrate that the ANTI-FLAG M2 plate allows for the rapid and quantitative determination of phosphorylation of FLAG-MAPK directly from stimulated, transfected cell lysate. Captured, phosphorylated FLAG-MAPK retains catalytic activity as demonstrated by the phosphorylation of Elk-1 in the same well. Furthermore, phosphorylation of captured FLAG-MAPK by the upstream kinases can be observed directly on the plate. These assays are sensitive, specific, and suitable for handling multiple samples. Thus, the ANTI-FLAG M2 plate forms the basis of a high-throughput screening platform in kinase analysis.
abstract_unstemmed	We have developed a rapid, sensitive, and quantitative 96-well microplate-based nonradioactive immunoprecipitation/kinase assay to evaluate mitogen-activated protein kinase (MAPK) activity. Three quantitative nonradioactive imunoprecipitation/kinase assays of MAPK were demonstrated on a 96-well microplate coated with ANTI-FLAG® M2 antibody (ANTI-FLAG M2 plate): (i) the capture of phosphorylated FLAG®-tagged MAPK fusion protein (FLAG-MAPK) from phorbol esters-stimulated, FLAG-MAPK-transfected COS-7 cells, coupled with a very sensitive ELISA procedure to quantitate the level of phosphorylation of FLAG-MAPK; (ii) the in vitro kinase reaction of FLAG-MAPK activity with a substrate and ATP in the same well used to captured the phosphorylated FLAG-MAPK; and (iii) the in vitro kinase reaction of captured non-activated FLAG-MAPK by its upstream kinase from phorbol 12-myristate 13-acetate (PMA)-stimulated COS-7 cells. These results demonstrate that the ANTI-FLAG M2 plate allows for the rapid and quantitative determination of phosphorylation of FLAG-MAPK directly from stimulated, transfected cell lysate. Captured, phosphorylated FLAG-MAPK retains catalytic activity as demonstrated by the phosphorylation of Elk-1 in the same well. Furthermore, phosphorylation of captured FLAG-MAPK by the upstream kinases can be observed directly on the plate. These assays are sensitive, specific, and suitable for handling multiple samples. Thus, the ANTI-FLAG M2 plate forms the basis of a high-throughput screening platform in kinase analysis.
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container_issue	2
title_short	Nonradioactive Assay of FLAG®-Tagged MAPK Using ANTI-FLAG® Antibody-Coated Multiwell Plates
url	https://doi.org/10.2144/02322pf02 https://doaj.org/article/7e10e6cd099c46f4bb32c8ba528a9bd3 https://www.future-science.com/doi/10.2144/02322pf02 https://doaj.org/toc/0736-6205 https://doaj.org/toc/1940-9818
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up_date	2024-07-03T19:32:49.091Z
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