Western blot analysis of extracts from HeLa cells, mock transfected or transfected with mouse WWOX, and from MCF-7 and rat brain using WWOX 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 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
WWOX Antibody detects endogenous levels of total WWOX protein.Species Reactivity:
Human, Mouse, RatSpecies predicted to react based on 100% sequence homology:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Thr103 of WWOX. Antibodies were purified by peptide affinity chromatography.
The WWOX (WW domain-containing oxidoreductase) gene encodes a protein with two WW domains followed by a short-chain dehydrogenase domain that was identified from a genomic region 16q23 of high instability, FRA16D (1,2). The mouse homolog, termed Wox1, was found to enhance TNFα-mediated apoptosis (3). The WWOX gene is disrupted in a many cancer types by deletions or translocation which has revealed a tumor suppressor function (4-7). In contrast, high levels of WWOX have been shown in shown in premetastic cancers, including breast and prostate (8-10). Stress stimuli can induce tyrosine phosphorylation within the first WW domain (Tyr33), followed by nuclear translocation and binding to and stabilizing the p53 tumor suppressor protein (11). WWOX and p53 can induce apoptosis in a synergistic manner. Tyrosine phosphorylation and nuclear translocation of WWOX has been implicated in the progression of cancers to metastatic states (10).
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