|H M R||Endogenous||60, 50||Rabbit IgG|
Western blot analysis of extracts from MKN-45 cells treated with λ-phosphatase or Forskolin #3828 (30 μM, 20 min) using Phospho-CaMKII (Thr286) (D21E4) Rabbit mAb (upper) and CaMKII-α (D10C11) Rabbit mAb #11945 (lower).Learn more about how we get our images.
Western blot analysis of extracts from rat brain, mouse brain and human cerebellum using Phospho-CaMKII (Thr286) (D21E4) Rabbit mAb.Learn more about how we get our images.
Western blot analysis of extracts from 293T cells, untransfected or transfected with specific CamKII proteins as indicated, using Phospho-CamKII (Thr286) (D21E4) Rabbit mAb. Antibody phosphospecificity was verified by preincubating the antibody in the absence of a peptide (-) or with either CamKII-β (Thr287) phosphopeptide (+) or CamKII-β (Thr287) non-phosphopeptide (+) prior to incubating the membrane.Learn more about how we get our images.
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 datasheet or 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 November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised October 2016
Protocol Id: 10
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
Phospho-CaMKII (Thr286) (D21E4) Rabbit mAb recognizes endogenous levels of CamKII-α protein only when phosphorylated at Thr286. This antibody also recognizes endogenous levels of CamKII-β and CamKII-γ protein only when phosphorylated at Thr287.
Human, Mouse, Rat
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr287 of human CamKII-β protein.
CaMKII is an important member of the calcium/calmodulin-activated protein kinase family, functioning in neural synaptic stimulation and T cell receptor signaling (1,2). CaMKII has catalytic and regulatory domains. Ca2+/calmodulin binding to the CaMKII regulatory domain relieves autoinhibition and activates the kinase (3). The activated CaMKII further autophosphorylates at Thr286 to render the kinase constitutively active (3). The threonine phosphorylation state of CaMKII can be regulated through PP1/PKA. PP1 (protein phosphatase 1) dephosphorylates phospho-CaMKII at Thr286. PKA (protein kinase A) prevents phospho-CaMKII (Thr286) dephosphorylation through an inhibitory effect on PP1 (4).
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