A rapid and sensitive method for detection and quantification of calcineurin and calmodulin-binding proteins using biotinylated calmodulin.
Purified bovine brain calmodulin was biotinylated with biotinyl-epsilon-aminocaproic acid N-hydroxysuccinimide. Biotinylated calmodulin was used to detect and quantify calmodulin-binding proteins following both protein blotting and slot-blot procedures by using alkaline phosphatase or peroxidase coupled to avidin. When purified bovine brain calcineurin, a calmodulin-dependent protein phosphatase, was immobilized on nitrocellulose slot blots, biotinylated calmodulin bound in a calcium-dependent saturable manner; these blots were then quantified by densitometry. Biotinylated calmodulin was able to detect as little as 10 ng of calcineurin, and the binding was competitively inhibited by addition of either native calmodulin or trifluoperazine. When biotinylated calmodulin was used to probe protein blots of crude brain cytosol and membrane preparations after gel electrophoresis, only protein bands characteristic of known calmodulin-binding proteins (i.e., calmodulin-dependent protein kinase, calcineurin, spectrin) were detected with avidin-peroxidase or avidin-alkaline phosphatase procedures. Purified calcineurin was subjected to one- and two-dimensional gel electrophoresis and protein blotting; as expected, only the 61-kDa calmodulin-binding subunit was detected. When the two-dimensional protein blot was incubated with biotinylated calmodulin and detected with avidin-alkaline phosphatase, several apparent forms of the 61-kDa catalytic subunit were detected, consistent with isozymic species of the enzyme. The results of these studies suggest that biotinylated calmodulin can be used as a simple, sensitive, and quantifiable probe for the study of calmodulin-binding proteins.
D000818:Animals; D001710:Biotin; D002148:Calmodulin-Binding Proteins / Q000032:analysis*; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing
D002148:Calmodulin-Binding Proteins; D001710:Biotin