Detection of calmodulin-binding proteins using a 32P-labeled GST-calmodulin fusion protein and a novel renaturation protocol
To identify calmodulin-binding proteins in cellular extracts and tissue homogenates and to analyze purified calmodulin target proteins, overlay procedures using 125I-calmodulin or, more recently, nonradioactive biotinylated calmodulin have been widely used. Here we describe a rapid, alternative meth...
| Veröffentlicht in: | BioTechniques. - 1991. - 21(1996), 2 vom: 23. Aug., Seite 292-6 |
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| Weitere Verfasser: | , |
| Format: | Aufsatz |
| Sprache: | English |
| Veröffentlicht: |
1996
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| Zugriff auf das übergeordnete Werk: | BioTechniques |
| Schlagworte: | Journal Article Research Support, Non-U.S. Gov't Calmodulin Calmodulin-Binding Proteins Parvalbumins Phosphorus Radioisotopes Recombinant Fusion Proteins Glutathione Transferase EC 2.5.1.18 Cyclic AMP-Dependent Protein Kinases mehr... |
| Zusammenfassung: | To identify calmodulin-binding proteins in cellular extracts and tissue homogenates and to analyze purified calmodulin target proteins, overlay procedures using 125I-calmodulin or, more recently, nonradioactive biotinylated calmodulin have been widely used. Here we describe a rapid, alternative method for detecting calmodulin-binding proteins with a 32P-labeled calmodulin probe generated as a glutathione-S-transferase (GST)-fusion protein. We used a modified pGEX-2TK vector, which contains the flag epitope and the consensus sequence R-R-A-S, that can be phosphorylated by the cAMP-dependent protein kinase A. The fusion protein is easily purified from bacterial bysates by affinity chromatography using glutathione-Sepharose 4B beads. Phosphorylation of GST-calmodulin is performed directly on the beads and, after elution with reduced glutathione, the labeled calmodulin probe can be used for overlay experiments. We also describe a rapid renaturation protocol that enhances the signal for some but not all calmodulin-binding proteins and is used after the proteins have been transferred to nitrocellulose filters. Furthermore, we have compared the specificity and sensitivity of the 32P-labeled GST-calmodulin overlay with those of 125I-calmodulin and biotinylated calmodulin, clearly indicating that our newly developed protocol is a suitable alternative to conventionally used calmodulin overlay procedures |
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| Beschreibung: | Date Completed 03.04.1997 Date Revised 28.09.2018 published: Print Citation Status MEDLINE |
| ISSN: | 1940-9818 |