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AlphaLISA SureFire Biotin-Free Human Androgen Receptor (Variant 7/AR-V7) Detection Kit, 500 Assay Points

The AlphaLISA™ SureFire® Biotin-Free Human Androgen Receptor (Variant 7/AR-V7) assay is a sandwich immunoassay for quantitative detection of Androgen Receptor Variant 7 (AR-V7) in cellular lysates using Alpha Technology.

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Feature	Specification
Application	Cell Signaling
Protocol Time	2h at RT
Sample Volume	10 µL

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Part #:

ASBF-TARV7-A-HV

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USD 737.00

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ASBF-TARV7-A500

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USD 2,490.00

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ASBF-TARV7-A10K

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USD 14,982.00

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ASBF-TARV7-A50K

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USD 47,624.00

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For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption and disposal requirements under European REACH regulations (EC 1907/2006).

Product information

Overview

Androgen Receptor splice variant 7 (AR-V7) is a constitutively active truncated isoform of AR that lacks the C-terminal ligand-binding domain (LBD) due to alternative splicing incorporating cryptic exon 3b. Because AR-V7 lacks the LBD, it is insensitive to androgen deprivation and is not inhibited by enzalutamide, apalutamide, or other LBD-targeting antiandrogens, rendering it a major driver of treatment resistance in castration-resistant prostate cancer (CRPC). AR-V7 retains the NTD and DBD, allowing it to constitutively translocate to the nucleus, bind AREs, and activate a transcriptional program that overlaps with but is distinct from AR-FL-driven gene expression. Detection of AR-V7 in circulating tumor cells (CTCs) has been validated as a predictive biomarker for resistance to enzalutamide and apalutamide, guiding clinical treatment decisions toward taxane-based chemotherapy in AR-V7-positive CRPC patients. AR-V7 specific reagents are essential tools for both clinical biomarker research and mechanistic studies of androgen-independent prostate cancer progression.

The AlphaLISA SureFire Biotin-Free Human Androgen Receptor (Variant 7/AR-V7) Detection Kit is a sandwich immunoassay for the quantitative detection of androgen receptor (Variant 7/AR-V7) in cellular lysates, using Alpha Technology.

Formats:

The HV (high volume) kit contains reagents to run 100 wells in 96-well format, using a 60 μL reaction volume.
The 500-point kit contains enough reagents to run 500 wells in 384-well format, using a 20 μL reaction volume.
The 10,000-point kit contains enough reagents to run 10,000 wells in 384-well format, using a 20 μL reaction volume.
The 50,000-point kit contains enough reagents to run 50,000 wells in 384-well format, using a 20 μL reaction volume.

AlphaLISA SureFire Biotin Free kits are compatible with:

Cell and tissue lysates
Antibody modulators
Biotherapeutic antibodies
Biotin rich samples

AlphaLISA SureFire Biotin Free kits can be used for:

Cellular kinase assays
Receptor activation studies
High-throughput screening for preclinical studies

How it works

Total-AlphaLISA SureFire Biotin Free assay principle

The Total-AlphaLISA SureFire Biotin Free assay measures the expression level of a target protein in a biological sample (e.g. cell lysate).

The Total-AlphaLISA SureFire Biotin Free assay uses two antibodies which recognize two different distal epitopes on the target protein. AlphaLISA assays require two bead types: Acceptor and Donor Beads. Acceptor Beads are coated with a proprietary CaptSure™ agent to specifically immobilize the assay specific antibody, labeled with a CaptSure tag. Donor Beads are coated with a proprietary CaptSure3 agent to capture one of the detection antibodies, which is labeled with the CaptSure3 tag. In the presence of target protein, the two antibodies bring the Donor and Acceptor Beads in close proximity whereby the singlet oxygen transfers energy to excite the Acceptor Bead, allowing for the generation of a luminescent Alpha signal. The amount of light emission is directly proportional to the quantity of protein present in the sample.

Total-AlphaLISA SureFire Ultra Biotin Free assay principle

Total-AlphaLISA SureFire Biotin Free two-plate assay protocol

The two-plate protocol involves culturing and treating the cells in a 96-well plate before lysis, then transferring lysates into a 384-well OptiPlate™ plate before the addition of Total-AlphaLISA SureFire Biotin Free detection reagents. This protocol enables cell viability and confluence to be monitored. In addition, lysates from a single well can be used to measure multiple targets.

2 plates assay protocol alphalisa surefire ultra biotin free total assay

Total-AlphaLISA SureFire Biotin Free one-plate assay protocol

Detection of Total target protein with AlphaLISA SureFire Biotin Free reagents can be performed in a single plate used for culturing, treatment, and lysis. No washing steps are required. This HTS designed protocol allows for miniaturization while maintaining robust AlphaLISA SureFire quality.

1 plate assay protocol alphalisa surefire ultra biotin free total assay

Assay validation

AR-V7 degradation in 22Rv1 cells

22Rv1 cells were seeded in a 96-well plate (60,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO₂. Cells were treated with increasing concentrations of AR Degrader-8 for 24 hours.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Lysates were further diluted then AR-V7 and ERK Total levels were evaluated using AlphaLISA SureFire Biotin Free assays. For the detection step, 10 µL of cell lysate (approximately 1,200 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

AR Degrader-8 induced a dose-dependent decrease in AR-V7 with no significant changes in ERK Total levels.

Pharmacological Validation (Degradation) of AR-V7 Total assay

Assay versatility

Differential expression of AR-V7 in endogenous cell lines

Adherent cells were grown to confluency in a T175 flask in complete medium and lysed with Lysis Buffer at a density of 0.5 x 10⁶ cells/mL. Suspension cells were harvested, spun down and lysed with Lysis Buffer at a density of 1.6 x 10⁶ cells/mL.

AR-V7 levels were evaluated using the AlphaLISA SureFire Biotin Free assay. For the detection step, 10 µL of cell lysate (approximately 5,000 adherent and 16,000 suspension cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor Mix and incubated for 1 hour at RT. Finally, 5 µL of Donor Mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

As expected, AR-V7 expression was only detected in 22Rv1 cells, and to a lesser extent in LNCaP cells.

Assay sensitivity

Assay sensitivity - cell lysate dilution

Cell lysate was prepared from 22Rv1 cells cultured to confluence in a T175 flask and lysed with 8 mL of Lysis Buffer for 10 minutes at RT with shaking.

Lysate was serially diluted in Lysis Buffer and AR-V7 levels were evaluated using the AlphaLISA SureFire Biotin Free assay. For the detection step, 10 µL of lysate was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at room temperature. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

Approximate number of cells per datapoint is indicated. The dotted line represents assay background. The assay can detect AR-V7 expression in less than 200 cells.

Specifications

Other specifications

Application	Cell Signaling
Automation Compatible	Yes
Brand	AlphaLISA SureFire Biotin-Free
Detection Modality	Alpha
Product Group	Kit
Protocol Time	2h at RT
Sample Volume	10 µL
Shipping Conditions	Shipped in Blue Ice
Target	Androgen
Target Class	Phosphoproteins
Target Species	Human
Technology	Alpha
Therapeutic Area	Oncology
Unit Size	500 Assay Points