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 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.
Fischer
R
R
University Zürich-Irchel, Switzerland.
Wei
Y
Y
Berchtold
M
M
D000818:Animals; D001483:Base Sequence; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000032:analysis* / Q000378:metabolism; D002846:Chromatography, Affinity; D016384:Consensus Sequence; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000032:analysis / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009695:Nucleic Acid Renaturation; D010320:Parvalbumins / Q000235:genetics; D010761:Phosphorus Radioisotopes / Q000032:analysis*; D010766:Phosphorylation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012680:Sensitivity and Specificity
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D010320:Parvalbumins; D010761:Phosphorus Radioisotopes; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D017868:Cyclic AMP-Dependent Protein Kinases; D005978:Glutathione