Western blot analysis of extracts from COS-1 cells expressing wild-type or mutant ER alpha, stimulated with beta-estradiol (100 nM) and EGF (100 ng/ml) for 30 minutes, using Phospho-Estrogen Receptor alpha (Ser104/106) Antibody (upper) or control ER alpha Antibody #2512 (lower). (Cell lysates provided by Dr. Simak Ali, Hammersmith Hospital, London.)Learn more about how we get our images
Western blot analysis of extracts from serum-starved MCF7 cells, untreated, treated with β-estradiol, or treated with β-estradiol and heregulin, using Phospho-Estrogen Receptor α (Ser104/106) Antibody. A non-specific band is detected at 90 kDa.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.
Phospho-Estrogen Receptor alpha (Ser104/106) Antibody detects endogenous levels of ER alpha only when phosphorylated at Ser104/106. It does not cross-react with the phosphorylated ER isoform beta.
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding serine 104/106 of human ER alpha. Antibodies are purified by protein A and peptide affinity chromatography.
Estrogen receptor α (ERα), a member of the steroid receptor superfamily, contains highly conserved DNA binding and ligand binding domains (1). Through its estrogen-independent and estrogen-dependent activation domains (AF-1 and AF-2, respectively), ERα regulates transcription by recruiting coactivator proteins and interacting with general transcriptional machinery (2). Phosphorylation at multiple sites provides an important mechanism to regulate ERα activity (3-5). Ser104, 106, 118, and 167 are located in the amino-terminal transcription activation function domain AF-1, and phosphorylation of these serine residues plays an important role in regulating ERα activity. Ser118 may be the substrate of the transcription regulatory kinase CDK7 (5). Ser167 may be phosphorylated by p90RSK and Akt (4,6). According to the research literature, phosphorylation at Ser167 may confer tamoxifen resistance in breast cancer patients (4).
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