Western blot analysis of extracts from Hep G2 cells, untreated (-) or treated with λ-phosphatase (+), using Phospho-HNF1α (Ser247) Antibody (upper) and HNF1α (D7Z2Q) Rabbit mAb #89670 (lower).Learn more about how we get our images.
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing myc-tagged human wild-type HNF1α (hHNF1α-wt-6xMyc; +) or myc-tagged human mutant HNF1α S247A (hHNF1α-S247A-6xMyc; +), using Phospho-HNF1α (Ser247) Antibody (upper), HNF1α (D7Z2Q) Rabbit mAb #89670 (middle), and β-Tubulin (D2N5G) Rabbit mAb #15115 (lower).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.
HNF1α (Ser247) Antibody recognizes endogenous levels of HNF1α protein only when phosphorylated at Ser247.
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ser247 of human HNF1α protein. Antibodies are purified by protein A and peptide affinity chromatography.
Hepatocyte nuclear factor 1α (HNF1α, also known as TCF1 or MODY3) is a transcription factor that plays a role in the tissue-specific regulation of liver gene expression (1). Research has shown that heterogeneous mutations of HNF1α are linked to maturity onset diabetes of the young (MODY) (2). Recent studies indicate that increased concentrations of free fatty acids can reduce the expression of FoxA2/HNF3β and HNF1α in pancreatic β-cells and lead to their nuclear exclusion, resulting in symptoms of several metabolic syndromes (3).
HNF1α is inhibited through Akt2-mediated phosphorylation at Ser247 and is a negative regulator of PPARγ gene transcription. Research studies have shown that inhibition of PPARγ is beneficial in steatosis-associated liver cancer in mouse models (4).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. Tween is a registered trademark of ICI Americas, Inc.
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