Western blot analysis of extracts from 293 cells, either untransfected or transfected with DYKDDDDK-tagged Rev-erbα expression constructs, using Phospho-Rev-erbα (Ser55/Ser59) Antibody (upper) or DYKDDDDK Tag Antibody (Binds to same epitope as Sigma's Anti-FLAG® M2 Antibody) #2368 (lower). The phospho-specificity of the antibody was verified by transfecting cells with a mutant DYKDDDDK-Rev-erbα (Ser55/59Ala) expression construct and by incubating 293/Rev-erbα (WT) extracts with (+) or without (-) λ phosphatase.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 and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
Phospho-Rev-erbα (Ser55/59) Antibody detects transfected levels of Rev-erbα protein when phosphorylated at Ser55/59. The antibody does not cross-react with other nuclear receptor proteins, including Rev-erbβ.
Mouse, Rat, Bovine
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser55 and Ser59 of human Rev-erbα. Antibodies are purified by protein A and peptide affinity chromatography.
Reverse orientation c-erbA gene α (Rev-erbα, EAR-1, or NR1D1) is a widely expressed member of the orphan nuclear receptor family of proteins (1). Rev-erbα is highly expressed in adipose tissue, skeletal muscle, brain and liver, and regulates cellular proliferation and differentiation. Expression increases during differentiation in adipocytes and ectopic expression of Rev-erbα potentiates the adipocyte differentiation of 3T3-L1 cells (2). In addition, expression oscillates with circadian rhythm in liver cells and Rev-erbα regulates expression of BMAL1, ApoA-I and ApoC-III, all key regulators of circadian rhythm (3-7). Phosphorylation of Rev-erbα Ser55 and Ser59 by GSK-3β appears to stabilize Rev-erbα protein levels and is important for synchronizing and maintaining the circadian clock (8). Rev-erbα also regulates inflammation by targeting the NF-κB responsive genes IL-6 and COX-2 (9). Rev-erbα lacks the activation function 2 domain required for ligand-dependent activation of transcription by other members of the nuclear receptor family; thus it behaves as a constitutive repressor protein, recruiting the nuclear receptor co-repressor (N-CoR)/HDAC3 complex to target genes to repress transcription (10).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. Anti-FLAG is a registered trademark of Sigma-Aldrich Biotechnology.
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