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...
Saved in:
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Taylor & Francis Group
2002-02-01
|
| Series: | BioTechniques |
| Online Access: | https://www.future-science.com/doi/10.2144/02322pf02 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1850152917549449216 |
|---|---|
| author | L. Zhang S. Uder T. Juehne B. Brizzard K. Song |
| author_facet | L. Zhang S. Uder T. Juehne B. Brizzard K. Song |
| author_sort | L. Zhang |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-7e10e6cd099c46f4bb32c8ba528a9bd3 |
| institution | OA Journals |
| issn | 0736-6205 1940-9818 |
| language | English |
| publishDate | 2002-02-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | BioTechniques |
| spelling | doaj-art-7e10e6cd099c46f4bb32c8ba528a9bd32025-08-20T02:25:51ZengTaylor & Francis GroupBioTechniques0736-62051940-98182002-02-0132244244710.2144/02322pf02Nonradioactive Assay of FLAG®-Tagged MAPK Using ANTI-FLAG® Antibody-Coated Multiwell PlatesL. Zhang0S. Uder1T. Juehne2B. Brizzard3K. Song41Sigma-Aldrich, St. Louis, MO, USA1Sigma-Aldrich, St. Louis, MO, USA1Sigma-Aldrich, St. Louis, MO, USA1Sigma-Aldrich, St. Louis, MO, USA1Sigma-Aldrich, St. Louis, MO, USAWe 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.https://www.future-science.com/doi/10.2144/02322pf02 |
| spellingShingle | L. Zhang S. Uder T. Juehne B. Brizzard K. Song Nonradioactive Assay of FLAG®-Tagged MAPK Using ANTI-FLAG® Antibody-Coated Multiwell Plates BioTechniques |
| title | Nonradioactive Assay of FLAG®-Tagged MAPK Using ANTI-FLAG® Antibody-Coated Multiwell Plates |
| title_full | Nonradioactive Assay of FLAG®-Tagged MAPK Using ANTI-FLAG® Antibody-Coated Multiwell Plates |
| title_fullStr | Nonradioactive Assay of FLAG®-Tagged MAPK Using ANTI-FLAG® Antibody-Coated Multiwell Plates |
| title_full_unstemmed | Nonradioactive Assay of FLAG®-Tagged MAPK Using ANTI-FLAG® Antibody-Coated Multiwell Plates |
| title_short | Nonradioactive Assay of FLAG®-Tagged MAPK Using ANTI-FLAG® Antibody-Coated Multiwell Plates |
| title_sort | nonradioactive assay of flag r tagged mapk using anti flag r antibody coated multiwell plates |
| url | https://www.future-science.com/doi/10.2144/02322pf02 |
| work_keys_str_mv | AT lzhang nonradioactiveassayofflagtaggedmapkusingantiflagantibodycoatedmultiwellplates AT suder nonradioactiveassayofflagtaggedmapkusingantiflagantibodycoatedmultiwellplates AT tjuehne nonradioactiveassayofflagtaggedmapkusingantiflagantibodycoatedmultiwellplates AT bbrizzard nonradioactiveassayofflagtaggedmapkusingantiflagantibodycoatedmultiwellplates AT ksong nonradioactiveassayofflagtaggedmapkusingantiflagantibodycoatedmultiwellplates |