Western blot analysis of extracts from LNCaP and SNB-19 cells, and mouse brain, untreated (-) or treated with λ phosphatase (+), using Phospho-CaMKK2 (Ser495) Antibody (upper), CaMKK2 (D8D4D) Rabbit mAb #16810 (middle), and β-Actin (D6A8) Rabbit mAb #8457 (lower).
|MW (kDa)||68, 70|
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Protocol Id: 10
Phospho-CaMKK2 (Ser495) Antibody recognizes endogenous levels of CaMKK2 protein only when phosphorylated at Ser495. Bands of unknown origin are observed at 42 kDa and 25 kDa.
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser495 of human CaMKK2 protein. Antibodies are purified by protein A and peptide affinity chromatography.
Calcium/Calmodulin-dependent Protein Kinase Kinase 2 (CaMKK2) is a member of the CaMK family that contains a central Ser/Thr kinase domain followed by a regulatory domain consisting of overlapping autoinhibitory and CaM-binding regions (1). CaMKK2 can be distinguished from other CaMK family members by the presence of a unique Pro/Arg/Gly-rich insert following the ATP-binding domain (2). CaMKK2 phosphorylates CaMKI at Thr177 and CaMKIV at Thr200 (3). CaMKK2 also phosphorylates AMPKα in response to calcium (4). CaMKK2 has been implicated in long-term memory formation (5) and adipocyte development (6). CaMKK2 is phosphorylated at Ser511 by death-associated protein kinase (DAPK) in a signaling cascade thought to be involved in neuronal death (7).
Multiple CaMKK2 phosphorylation sites have been identified, including Ser495, and C-terminal phosphorylation of the CaMKK2 autoinhibitory region may regulate its function and localization (8).
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