Western blot analysis of extracts of COS cells untransfected (lane 1), or transfected with wild-type mouse C/EBPalpha (lane 2), S21A (lane 3), and S21D (lane 4) mutants, using Phospho-C/EBPalpha (Ser21) Antibody (upper) or C/EBPalpha Antibody (lower). (Provided by Dr. Hanna Radomska, Beth Israel Deaconess Medical Center, Boston, MA).
Western blot analysis of extracts from mouse adipocytes treated with insulin for the indicated times, using Phospho-C/EBPalpha (Ser21) Antibody.
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-C/EBPα (Ser21) Antibody detects endogenous levels of C/EBPα only when phosphorylated at serine 21. This antibody does not cross-react with other phosphorylated C/EBP isoforms.
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser21 of human C/EBPα. Antibodies are purified by protein A and peptide affinity chromatography.
CCAAT/enhancer-binding proteins (C/EBPs) are a family of transcription factors that are critical for cellular differentiation, terminal function, and inflammatory response (1). Six members of the family have been characterized (C/EBPα, β, δ, γ, ε, and ζ) and are distributed in a variety of tissues (1). Translation from alternative start codons results in two isoforms of C/EBPα (p42 and p30), which are both strong transcriptional activators (2). It has been reported that insulin and insulin-like growth factor-I stimulate the dephosphorylation of C/EBPα, which may play a key role in insulin-induced repression of GLUT4 transcription (3). Phosphorylation of C/EBPα at Thr222, Thr226, and Ser230 by GSK-3 seems to be required for adipogenesis (4).
Explore pathways + proteins related to this product.
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