Kinase Mobility Shift Assay (KiMSA) for Assessing Protein Kinase A Activity
The cAMP-dependent protein kinase (PKA) is one of the most extensively distributed kinases among intracellular signal cascades, with a pivotal role in the regulation of various processes, including the capacitation of sperm cells. Traditional assessments of PKA activity rely on the utilization of [γ...
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Bio-protocol LLC
2025-07-01
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| author | Analia Novero Tomás Steeman Catalina Curcio Lara Buccolini Andres Binolfi Diego Krapf Mariano Buffone Dario Krapf Cintia Stival |
| author_facet | Analia Novero Tomás Steeman Catalina Curcio Lara Buccolini Andres Binolfi Diego Krapf Mariano Buffone Dario Krapf Cintia Stival |
| author_sort | Analia Novero |
| collection | DOAJ |
| description | The cAMP-dependent protein kinase (PKA) is one of the most extensively distributed kinases among intracellular signal cascades, with a pivotal role in the regulation of various processes, including the capacitation of sperm cells. Traditional assessments of PKA activity rely on the utilization of [γ-32P] ATP and the Kemptide peptide as a substrate. This strategy presents several major drawbacks, including high costs and health risks derived from the manipulation of radioactive isotopes. In this work, we introduce an enhanced non-radioactive assay to quantify PKA activity, termed kinase mobility shift assay (KiMSA), based on the use of a fluorescent-labeled Kemptide (Kemptide-FITC). Once the kinase reaction is terminated, the products can be easily resolved through electrophoresis on an agarose gel and quantified by fluorescence densitometry. We show that KiMSA is suitable for isolated PKA as well as for the enzyme in cell extracts. In addition, it enables quantification of PKA activity during the progression of mouse sperm capacitation. Furthermore, the assay enables monitoring the inhibition of PKA with pharmacological inhibitors in live cells. Therefore, the experimental and optimal assay conditions are set so that KiMSA can be used to assess in vitro as well as in vivo PKA activity in sperm cells. Finally, this method allows for measurement of cAMP concentrations, rendering a versatile technique for the study of cAMP/PKA pathways. |
| format | Article |
| id | doaj-art-5ae91900d41b4d02bdaec31f3bd0c01e |
| institution | OA Journals |
| issn | 2331-8325 |
| language | English |
| publishDate | 2025-07-01 |
| publisher | Bio-protocol LLC |
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| spelling | doaj-art-5ae91900d41b4d02bdaec31f3bd0c01e2025-08-20T02:37:20ZengBio-protocol LLCBio-Protocol2331-83252025-07-01151310.21769/BioProtoc.5366Kinase Mobility Shift Assay (KiMSA) for Assessing Protein Kinase A ActivityAnalia Novero0Tomás Steeman1Catalina Curcio2Lara Buccolini3Andres Binolfi4Diego Krapf5Mariano Buffone6Dario Krapf7Cintia Stival8Cell Signal Transduction Networks Laboratory, IBR (CONICET-UNR), Rosario, ArgentinaCell Signal Transduction Networks Laboratory, IBR (CONICET-UNR), Rosario, ArgentinaCell Signal Transduction Networks Laboratory, IBR (CONICET-UNR), Rosario, ArgentinaCell Signal Transduction Networks Laboratory, IBR (CONICET-UNR), Rosario, ArgentinaCellular-Structural Biology Laboratory, IBR (CONICET-UNR), Rosario, ArgentinaDepartment of Electrical and Computer Engineering, Colorado State University, Fort Collins, CO, USAInstituto de Biología y Medicina Experimental (CONICET), Ciudad Autónoma de Buenos Aires, ArgentinaCell Signal Transduction Networks Laboratory, IBR (CONICET-UNR), Rosario, ArgentinaCell Signal Transduction Networks Laboratory, IBR (CONICET-UNR), Rosario, ArgentinaThe cAMP-dependent protein kinase (PKA) is one of the most extensively distributed kinases among intracellular signal cascades, with a pivotal role in the regulation of various processes, including the capacitation of sperm cells. Traditional assessments of PKA activity rely on the utilization of [γ-32P] ATP and the Kemptide peptide as a substrate. This strategy presents several major drawbacks, including high costs and health risks derived from the manipulation of radioactive isotopes. In this work, we introduce an enhanced non-radioactive assay to quantify PKA activity, termed kinase mobility shift assay (KiMSA), based on the use of a fluorescent-labeled Kemptide (Kemptide-FITC). Once the kinase reaction is terminated, the products can be easily resolved through electrophoresis on an agarose gel and quantified by fluorescence densitometry. We show that KiMSA is suitable for isolated PKA as well as for the enzyme in cell extracts. In addition, it enables quantification of PKA activity during the progression of mouse sperm capacitation. Furthermore, the assay enables monitoring the inhibition of PKA with pharmacological inhibitors in live cells. Therefore, the experimental and optimal assay conditions are set so that KiMSA can be used to assess in vitro as well as in vivo PKA activity in sperm cells. Finally, this method allows for measurement of cAMP concentrations, rendering a versatile technique for the study of cAMP/PKA pathways.https://bio-protocol.org/en/bpdetail?id=5366&type=0 |
| spellingShingle | Analia Novero Tomás Steeman Catalina Curcio Lara Buccolini Andres Binolfi Diego Krapf Mariano Buffone Dario Krapf Cintia Stival Kinase Mobility Shift Assay (KiMSA) for Assessing Protein Kinase A Activity Bio-Protocol |
| title | Kinase Mobility Shift Assay (KiMSA) for Assessing Protein Kinase A Activity |
| title_full | Kinase Mobility Shift Assay (KiMSA) for Assessing Protein Kinase A Activity |
| title_fullStr | Kinase Mobility Shift Assay (KiMSA) for Assessing Protein Kinase A Activity |
| title_full_unstemmed | Kinase Mobility Shift Assay (KiMSA) for Assessing Protein Kinase A Activity |
| title_short | Kinase Mobility Shift Assay (KiMSA) for Assessing Protein Kinase A Activity |
| title_sort | kinase mobility shift assay kimsa for assessing protein kinase a activity |
| url | https://bio-protocol.org/en/bpdetail?id=5366&type=0 |
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