Western blot analysis of extracts from various cell lines using Androgen Receptor (AR-V7 Specific) Antibody (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower). Signal corresponding to the androgen receptor V7 isoform is detected in 22Rv1 and VCAP cells (AR-V7-positive), but is not detected in LNCaP and DU 145 cells (AR-V7-negative), confirming specificity of the 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 nonfat dry milk, 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.
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 June 2016
Protocol Id: 263
Androgen Receptor (AR-V7 Specific) Antibody recognizes endogenous levels of total AR-V7 protein. This antibody does not cross-react with full-length AR protein.Species Reactivity:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu639 of human androgen receptor (V7 isoform) protein. Antibodies are purified by protein A and peptide affinity chromatography.
Androgen receptor (AR), a zinc finger transcription factor belonging to the nuclear receptor superfamily, is activated by phosphorylation and dimerization upon ligand binding (1). This promotes nuclear localization and binding of AR to androgen response elements in androgen target genes. Research studies have shown that AR plays a crucial role in several stages of male development and the progression of prostate cancer (2,3).
The AR3 or AR-V7 isoform, which lacks the typical ligand binding domain, is created through the alternative splicing of cryptic exons (4-5). AR-V7 is frequently expressed in castration-resistant prostate cancer (CRPC) and while dependent on the activity of the full-length androgen receptor (AR-FL), AR-V7 can activate a completely distinct transcriptional program (6-8). While enzalutamide and abiraterone have been beneficial in treating CRPC through the ligand binding domain of AR-FL, resistance in patients has been shown to be associated with AR-V7 detection in circulating tumor cells (9-12). Studies probing into mechanisms of overcoming this resistance are currently being explored and may help in stratifying patient populations for more personalized therapies (13-15).
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