Western blot analysis of extracts from HeLa cells, untransfected or transfected with human or mouse JMJD3, using JMJD3 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
JMJD3 Antibody detects transfected levels of JMJD3 protein.Species Reactivity:
Human, MouseSpecies predicted to react based on 100% sequence homology:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to the human JMJD3 protein. Antibodies are purified by protein A and peptide affinity chromatography.
The methylation state of lysine residues in histone proteins is a major determinant of the formation of active and inactive regions of the genome and is crucial for proper programming of the genome during development (1,2). Jumonji C (JmjC) domain-containing proteins represent the largest class of potential histone demethylase proteins (3). The JmjC domain can catalyze the demethylation of mono-, di-, and tri-methyl lysine residues via an oxidative reaction that requires iron and α-ketoglutarate (3). Based on homology, both humans and mice contain at least 30 such proteins, which can be divided into 7 separate families (3). The three members of the UTX/UTY family include the ubiquitously transcribed X chromosome tetratricopeptide repeat protein (UTX), the ubiquitously transcribed Y chromosome tetratricopeptide repeat protein (UTY) and JmjC domain-containing protein 3 (JMJD3) (3). This family of proteins has been shown to demethylate both di- and tri-methyl histone H3 Lys 27 (4-8). The UTX gene escapes X inactivation in females and is ubiquitously expressed (9). UTX functions to regulate HOX gene expression during development (4-6). JMJD3 functions to regulate gene expression in macrophages responding to various inflammatory stimuli and has been shown to be upregulated in prostate cancer (7,8). Both UTX and JMJD3 interact with mixed-lineage leukemia (MLL) complexes 2 and 3, both of which have been shown to methylate histone H3 at Lys4 (6,7). The UTY gene is expressed in most tissues in the male mouse (10).
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