Western blot analysis of extracts from HeLa cells, untransfected or transfected with a contruct overexpressing human WTX, using WTX (D38E5) Rabbit mAb.
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. 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
WTX (D38E5) Rabbit mAb detects transfected levels of total WTX protein.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with recombinant protein specific to the amino terminus of human WTX protein.
WTX (Wilms’ tumor gene on the X chromosome) is a developmentally regulated gene with a potentially important role in kidney development (1). Functional studies of WTX suggest that it acts as a tumor suppressor gene in renal cells by promoting β-catenin ubiquitination and degradation, thereby antagonizing WNT/β-catenin signaling (1,2). WTX is found to be inactivated in 30% of Wilms’ tumors, mostly by chromosomal deletion (3). Wilms’ tumor is a pediatric kidney cancer that arises from cells that fail to differentiate during kidney development (4). Inactivation of the WT1 tumor suppressor gene accounts for 10-15% of Wilms’ tumor cases (5). WTX has been shown to enhance WT1-mediated transactivation, suggesting a physiologically significant interaction between WT1 and WTX (6). WTX may be directly involved in the transcriptional regulation of cellular differentiation in the kidney through interactions with WT1 and other transcription factors (6).
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