Calmodulin-binding protein detection using a non-radiolabeled calmodulin fusion protein.
Calmodulin-binding proteins are involved in numerous cellular signaling pathways. The biotinylated-calmodulin overlay is a nonradioactive method widely used to detect calmodulin-binding proteins in tissue and cell samples. This method has several limitations; therefore, we developed a nonradioactive calmodulin-binding protein detection overlay using an S-tag-labeled calmodulin fusion protein. An expression system was used to generate a calmodulin fusion protein with an S-tag label, a 15 amino acid sequence that binds to a 105 amino acid S-protein. The S-protein is conjugated to horseradish peroxidase for final detection with a chemiluminescent substrate. The S-tag calmodulin was compared to purified calmodulin and biotinylated calmodulin in a calmodulin-dependent phosphodiesterase assay. The results of the calmodulin-dependent phosphodiesterase assay indicate that S-tag calmodulin induces higher phosphodiesterase activity than biotinylated calmodulin and lower activity than purified calmodulin. A comparison of the biotinylated and S-tag calmodulin overlay assays indicate that S-tag calmodulin is more sensitive than biotinylated calmodulin in the detection of calcineurin, a known calmodulin-binding protein. The overlay assay results also indicate that the S-tag calmodulin and biotinylated calmodulin detect similar calmodulin-binding proteins in colon epithelial cells. In conclusion, the S-tag calmodulin overlay assay is a consistent, sensitive, and rapid nonradioactive method to detect calmodulin-binding proteins.
Murray
G
G
University of Idaho, Moscow, ID, USA.
Marshall
M J
MJ
Trumble
W
W
Magnuson
B A
BA
D000818:Animals; D019921:Biotinylation; D019703:Calcineurin / Q000032:analysis / Q000378:metabolism; D002147:Calmodulin / Q000235:genetics* / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000032:analysis* / Q000378:metabolism; D002645:Chickens; D003106:Colon / Q000737:chemistry; D004847:Epithelial Cells / Q000737:chemistry; D006735:Horseradish Peroxidase; D006801:Humans; D008163:Luminescent Measurements; D008297:Male; D010957:Plasmids / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012680:Sensitivity and Specificity
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D011993:Recombinant Fusion Proteins; D006735:Horseradish Peroxidase; D019703:Calcineurin