Anti-CaMKII (phospho T286) antibody (ab32678)

Blocked with 5% milk for 1 hour at RT.

Incubated with primary antibody for 14 hours at 4°C in TBS-T20.

See Abreview

ab32678 (2µg/ml) staining CaMKII (phospho T286) in human Brain:cortex:frontal-lateral using an automated system (DAKO Autostainer Plus). Using this protocol there is strong staining of nuclear/cytoplasmic compartments within the stellate cells and the myelinated fibres of white matter region .
Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer EDTA pH 9.0 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.

PC 12 cells were incubated at 37°C for 30 minutes with vehicle control (0 µM) and different concentrations of myricetin (ab120721). Increased expression of CaMKII (phospho T286) (ab32678) in PC 12 cells correlates with an increase in myricetin concentration, as described in literature.

Whole cell lysates were prepared with RIPA buffer (containing protease inhibitors and sodium orthovanadate), 20µg of each were loaded on the gel and the WB was run under reducing conditions. After transfer the membrane was blocked for an hour using 3% milk before being incubated with ab32678at 1/500 dilution and ab52476 at 1/500 dilution overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP (ab97051) at 1/10000 dilution and visualised using ECL development solution.