Western blot analysis of extracts from HuH-6 cells, mouse liver and rat liver using ChREBP Antibody.
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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 nonfat dry milk, 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.
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 June 2016
Protocol Id: 263
ChREBP Antibody recognizes endogenous levels of total ChREBP protein. Based on amino acid sequence comparisons, the antibody is predicted to detect all known isoforms of ChREBP.Species Reactivity:
Human, Mouse, Rat
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu308 of human ChREBP isoform α protein. The sequence surrounding this region is 100% conserved in all known isoforms of ChREBP. Antibodies are purified by protein A and peptide affinity chromatography.
Carbohydrate-responsive element-binding protein (ChREBP) is a glucose-responsive transcription factor that regulates glycolytic and lipogenic gene expression (1). High levels of glucose induce the transcriptional activity of ChREBP. ChREBP is most abundant in tissues of de novo lipogenesis and forms a heterotetramer with its binding partner MLX to bind to the promoter regions of its target genes (2). ChREBP regulates fatty acid synthesis, glucose homeostasis and insulin sensitivity in white adipose tissue. In white adipose tissue, ChREBP isoform α (ChREBP-α) activates the expression of the potent ChREBP isoform β (ChREBP-β) in a glucose-dependent manner. ChREBP-β expression levels predict insulin sensitivity in human white adipose tissue (1). In addition, research studies have shown that Akt2 induces the transcriptional activity of ChREBP-β to stimulate de novo lipogenesis in brown adipose tissue for fuel storage (3). Furthermore, ChREBP-β is a potent activator of lipogenesis in the liver (4).
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