title: Measuring CaMKII concentration in dendritic spines.
abstract: Here, we present a method for measuring the concentration of endogenous protein in cellular compartments. Importantly, the method is applicable to compartments such as dendritic spines with dimensions often close to the resolution limit of optical microscopy. To our knowledge, a method with such capabilities has not yet been described. The method utilizes overexpression of the protein of interest, which is tagged with fluorescent protein. This is followed by immunostaining of both overexpressed and endogenous proteins. Expression of a volume marker is also required. We applied this method to measure the concentration of Ca/calmodulin kinase II (CaMKII) in different cellular compartments of hippocampal pyramidal neurons. It was found that the concentrations of CaMKIIα subunits in cell bodies, proximal dendrites, and spines on these dendrites are 71, 46, and 103 μM, respectively. Considering the 3:1 ratio of α to β CaMKII subunits in the hippocampus, the concentrations of total (α+β) CaMKII subunits in these compartments are 94, 61, and 138 μM, respectively.
authors: Biology Department, Brandeis University, 415 South St., Waltham, MA 02454, United States. otmakhov@brandeis.edu
authors: Nikolai
authors: N
authors: Otmakhov
authors: John
authors: J
authors: Lisman
mesh_terms: D000818:Animals; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000032:analysis*; D049229:Dendritic Spines / Q000737:chemistry*; D018274:Electroporation; D005455:Fluorescent Antibody Technique / Q000379:methods*; D005456:Fluorescent Dyes; D007091:Image Processing, Computer-Assisted; D018613:Microscopy, Confocal; D017966:Pyramidal Cells / Q000737:chemistry; D014162:Transfection; D056969:Voltage-Sensitive Dye Imaging / Q000379:methods
chemical_list: D005456:Fluorescent Dyes; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2