Quantification of CaMKII isoforms in mouse brain

Olfactory Bulb, hippocampus, hypothalamus, cerebellum and brain stem were dissected from 8–12 week old C57Bl/6 mice. The dissected brain tissue was first sonicated in a buffer containing 10 mM Tris pH 8, 1 mM EDTA, and 1% SDS and then boiled for 5 min. Total protein concentrations were determined using a Pierce BCA protein assay kit (Thermo Scientific). Western-blot analysis after protein transfer onto PVDF membrane was done essentially as described previously^{12 (link),53 (link)–55 (link)}, using antibodies specific for CaMKIIβ (CBβ1) or CaMKIIα (CBα2). Chemo-luminescence detection was done using Super signal west femto ECL reagent (Thermo Scientific) for 3 minutes; images were acquired on a Fluorchem SP imager (Alpha Innotech). The 10% polyacrylamide SDS gels were precast criterion gels (BioRad); other percentage gels were made in house.