Western blot analysis of extracts from Jurkat cells, untreated or treated with TPA #9905 (40 nM) and calcium ionophore A23187 (2 μM) for 15 minutes, and λ-phosphatase plus calf intestinal phosphatase (CIP) as indicated, using Phospho-CARD11 (Ser652) Antibody (upper) and CARD11 (1D12) Rabbit mAb #4435 (lower).
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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 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
Phospho-CARD11 (Ser652) Antibody detects endogenous levels of CARD11 protein only when phosphorylated at Ser652.Species Reactivity:
Human, Mouse, RatSpecies predicted to react based on 100% sequence homology:
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to a region surrounding Ser652 of human CARD11 protein. Antibodies were purified by protein A and peptide affinity chromatography.
CARD11/Carma1/Bimp3 belongs to the MAGUK (membrane-associated guanylate kinase) family that typically function as molecular scaffolds in the assembly of multiprotein complexes (1,2). MAGUK family members contain an SH3 domain, a PDZ domain and a GuK domain homologous to guanylate kinase. In addition, CARD11 contains an amino-terminal CARD domain (caspase recruitment domain). This domain plays an important role in forming interactions with a number of proteins containing CARD domains that are involved in regulating apoptosis and NF-κB activation. CARD11 is predominately expressed in lymphocytes (1,2) and associates with the CARD domain of Bcl10. When overexpressed, CARD11 leads to the phosphorylation of Bcl10 and activation of NF-κB (1,2). CARD11 is constitutively associated with lipid rafts and is thought to function by recruiting Bcl10 and MALT1 and triggering the phosphorylation of IKKs (3,4). Several studies using the genetic disruption of CARD11 or dominant-negative mutations have demonstrated that it plays a critical role in NF-κB activation and lymphocyte signaling (4-7).
Phosphorylation at multiple sites within the central region of CARD11 regulates NF-κB activation (8-10).
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