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 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-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.Species Reactivity:
Human, MouseSpecies predicted to react based on 100% sequence homology:
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).
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