Western blot analysis of extracts from MCF7 and HeLa cells using C/EBPβ Antibody.
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|MW (kDa)||20 KD (LIP), 46 KD (LAP), 48 KD (Full Length)|
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
C/EBPβ Antibody recognizes endogenous levels of total C/EBPβ protein. This antibody does not cross-react with C/EBPα protein.Species Reactivity:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human C/EBPβ protein. Antibodies are purified by protein A and peptide affinity chromatography.
C/EBPβ is a member of the transcription factor family of CCAAT/enhancer-binding proteins (C/EBPs) that are critical for cellular differentiation and function (1). There are various N-terminally truncated C/EBPβ isoforms: full-length C/EBPβ, C/EBPβ-LAP (liver-enriched activator protein), and C/EBPβ-LIP (liver-enriched inhibitory protein). In triple-negative breast cancer cells, aerobic glycolysis was shown to control the expression of C/EBPβ-LAP, which in turn stimulates the expression of granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF). These factors are secreted from the cancer cells to promote myeloid-derived suppressor cell (MDSC) development in the tumor microenvironment, thereby suppressing anti-tumor immunity (2). In addition, research studies showed that decreased C/EBPβ-LIP expression delays age-related phenotypes in mice, suggesting a potential role for C/EBPβ-LIP in aging (3).
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