This antibody is specific for the ~50 kDa alpha CaM Kinase II subunit and the ~60 kDa beta CaM Kinase II subunit phosphorylated at Thr286 in Western blots. Immunolabeling is blocked by the phosphopeptide used as the antigen but not by the corresponding dephosphopeptide.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/1000. Predicted molecular weight: 50 kDa. Predicted molecular weight: ~50 kDa for the alpha subunit and ~60 kDa for the beta subunit of CaMKII.
Use a concentration of 2 µg/ml.
Use at an assay dependent concentration.
CaM-kinase II (CAMK2) is a prominent kinase in the central nervous system that may function in long-term potentiation and neurotransmitter release. Member of the NMDAR signaling complex in excitatory synapses it may regulate NMDAR-dependent potentiation of the AMPAR and synaptic plasticity.
Belongs to the protein kinase superfamily. CAMK Ser/Thr protein kinase family. CaMK subfamily. Contains 1 protein kinase domain.
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.
Western blot - Anti-CaMKII (phospho T286) antibody (ab32678)
Developed using the ECL technique
Performed under reducing conditions.
Predicted band size : 50 kDa
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.
Western blot - CaMKII (phospho T286) antibody (ab32678)
All lanes : Anti-CaMKII (phospho T286) antibody (ab32678) at 1/1000 dilution
Lane 1 : Rat brain cortex lysate. Lane 2 : Rat brain cortex lysate preincubated with lambda-phosphatase.
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