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| Product Name | Androgen Receptor (AR) ELISA Kit, Quantitative, Cell and Tissue Lysate |
| Catalog No. | TMTR-HMM-0083 |
| Description | Sandwich ELISA kit for quantitative detection of human androgen receptor (AR/NR3C4) protein in cell and tissue lysates. AR is a ligand-activated nuclear transcription factor and member of the steroid hormone receptor superfamily, playing a central role in prostate cancer development, progression, and resistance to androgen deprivation therapy (ADT). AR protein expression levels correlate with prostate cancer prognosis, metastatic potential, and response to anti-androgen therapeutics (enzalutamide, abiraterone, apalutamide, darolutamide). The assay uses a pre-coated 96-well plate with a capture antibody specific to the N-terminal domain (NTD) of human AR, and a detection antibody targeting the ligand-binding domain (LBD), providing specific detection of full-length AR (AR-FL) and clinically relevant AR splice variants (AR-V7, ARv567es) where both epitopes are present. The kit includes a recombinant AR protein standard, sample diluent, and all necessary reagents. |
| Intended Use | Quantitative measurement of androgen receptor protein in: prostate cancer cell lines (LNCaP, VCaP, 22Rv1, PC-3, DU145 — AR-positive and AR-negative controls); prostate tumor tissue lysates (fresh, frozen, or FFPE-derived protein extracts); circulating tumor cell (CTC) lysates; xenograft tumor lysates; and normal prostate tissue for research studies on prostate cancer biology, androgen signaling, and therapeutic response monitoring. |
| Principle / Technology | Sandwich ELISA: capture antibody (mouse monoclonal anti-AR NTD) adsorbed on microplate binds AR from the sample. After washing, biotinylated detection antibody (rabbit monoclonal anti-AR LBD) binds to a different AR epitope. Streptavidin-HRP binds to the detection antibody. TMB substrate is hydrolyzed by HRP to produce a colorimetric signal (A450) proportional to AR protein concentration. Quantitation against recombinant AR protein standard curve. |
| Detection Method | 1) Prepare lysates: homogenize cells/tissue in RIPA buffer with protease and phosphatase inhibitors; 2) Add 100 uL standard or sample to wells; incubate 2 h at RT with shaking; 3) Wash 4x; 4) Add 100 uL Detection Antibody; incubate 1 h at RT; 5) Wash 4x; 6) Add 100 uL Streptavidin-HRP; incubate 30 min at RT; 7) Wash 4x; 8) Add 100 uL TMB; incubate 15-30 min at RT in dark; 9) Add 100 uL Stop Solution; 10) Read A450; 11) Calculate AR concentration from 4-PL standard curve; 12) Normalize to total protein (BCA or Bradford). |
| Sample Type | Cell lysates: 10-100 ug total protein per well; tissue lysates: 20-200 ug total protein per well; samples in RIPA or modified RIPA buffer with protease inhibitors (PMSF, leupeptin, aprotinin) and phosphatase inhibitors (NaF, Na3VO4). |
| Performance Range / Specifications | Standard curve range: 0.078-5 ng/mL recombinant AR (typical); analytical sensitivity (LOD): <0.05 ng/mL; linear range: 0.16-5 ng/mL (R2 >0.99); AR detection in LNCaP cell lysate: approximately 20-50 ng AR/mg total protein; AR detection in AR-negative PC-3 cells: <0.1 ng/mg (background). |
| Sensitivity / LOD | Detection limit: <0.05 ng/mL recombinant AR (approximately 0.5 pM for 110 kDa protein); AR detectable in as few as 5,000 LNCaP cells (approximately 2 ug total protein). |
| Specificity | Specific for human androgen receptor: no cross-reactivity with glucocorticoid receptor (GR), progesterone receptor (PR), estrogen receptor alpha (ER-alpha), estrogen receptor beta (ER-beta), or mineralocorticoid receptor (MR) at 10 ng/mL. Detects full-length AR (AR-FL, ~110 kDa) and splice variants with intact NTD (AR-V7 and ARv567es confirmed). Does not detect AR splice variants lacking the NTD epitope. |
| Reaction Conditions / Protocol | Capture antibody incubation: 2 h RT; detection antibody: 1 h RT; streptavidin-HRP: 30 min RT; TMB: 15-30 min RT; total assay time: approximately 4-5 hours. |
| Components / Formulation | AR Antibody Coated 96-Well Microplate (12 x 8-well strips), Recombinant Human AR Standard (lyophilized, 1 vial), Biotinylated Anti-AR Detection Antibody (100x, 120 uL), Streptavidin-HRP (100x, 120 uL), Sample Diluent (25 mL), Standard Diluent (15 mL), Wash Buffer (20x, 50 mL), TMB Substrate (12 mL), Stop Solution (12 mL), Plate Sealers (4 sheets), Protocol. |
| Storage Conditions | Plate, Diluents, Wash Buffer, TMB, and Stop at 2-8 C; Standard, Detection Antibody, and Streptavidin-HRP at -20 C; protect from light. |
| Shelf Life | 12 months from date of manufacture. |
| Package Specifications | 96 determinations (one 96-well plate). |
| Product Form | Pre-coated strip plate; liquid reagents; lyophilized standard. |
| Quality Control | Each lot: standard curve R2 >0.99 (4-PL fit); LOD <0.05 ng/mL; intra-assay CV <8%; inter-assay CV <12%; spike recovery: 85-115% in LNCaP lysate matrix; specificity: no cross-reactivity with GR, PR, ER-alpha/beta, MR at 10 ng/mL; lot-to-lot consistency CV <15%. |
| Key Features | Quantitative AR sandwich ELISA; detects AR-FL and AR-V7; validated in prostate cancer cell lines; recombinant AR standard included; strip plate format; colorimetric TMB detection; sensitivity <0.05 ng/mL. |
| Purity | Anti-AR antibodies: >95% pure; recombinant AR standard: >90% pure by SDS-PAGE; all reagents: analytical grade. |
| Concentration | Recombinant AR standard: reconstitute to 5 ng/mL, serially dilute for 7-point standard curve; Detection Antibody and Streptavidin-HRP: 100x concentrate. |
| Activity / Unit Definition | Anti-AR NTD antibody affinity: Kd ~10^-10 M; anti-AR LBD antibody affinity: Kd ~10^-10 M; Streptavidin-HRP: >200 U/mg. |
| Molecular Weight | Full-length AR: approximately 110 kDa (919 amino acids); AR-V7 splice variant: approximately 75-80 kDa. |
| Source / Origin | Anti-AR antibodies: rabbit and mouse monoclonal (recombinant, animal-free production); recombinant AR standard: expressed in baculovirus-infected insect cells; HRP: plant-derived; all reagents: animal-free except as noted. |
| pH Range / Optimal pH | Sample Diluent pH 7.2-7.4; optimal binding pH 7.0-8.0; TMB pH ~4.0; Stop Solution pH <1.0. |
| Shipping Conditions | Cold pack (2-8 C); Standard and antibodies on dry ice recommended. |
| Expiration Date / Stability | 12 months at recommended conditions; reconstituted standard: 1 week at 2-8 C, 3 months at -80 C; plate strips in sealed pouch: 4 weeks at 2-8 C after opening. |
| Regulatory / Compliance | For research use only; not for diagnostic or therapeutic use. Not cleared by FDA or other regulatory bodies for clinical diagnostic use in prostate cancer management. |
| Compatibility | Samples must be lysed in RIPA or compatible lysis buffer; SDS concentration <0.05% in final sample dilution. DTT, beta-mercaptoethanol up to 1 mM do not interfere. Protease and phosphatase inhibitors recommended to prevent AR degradation. Compatible with tissue lysis methods: mechanical homogenization, sonication, freeze-thaw lysis. AR is susceptible to proteasomal degradation; process samples quickly on ice and add MG-132 (10 uM) or proteasome inhibitors if degradation is observed. |
| Recommended Buffer System | Sample Diluent: PBS pH 7.4, 1% BSA, 0.05% Tween-20; Standard Diluent: PBS pH 7.4, 1% BSA; Wash Buffer: PBS pH 7.4, 0.05% Tween-20. |
| Application Notes / Precautions | AR is a labile protein subject to rapid degradation. Lysates must be prepared fresh or from snap-frozen tissue/cell pellets stored at -80 C. Use protease inhibitor cocktail containing AEBSF/PMSF, leupeptin, aprotinin, and pepstatin, plus phosphatase inhibitors (NaF, Na3VO4, beta-glycerophosphate). AR expression is androgen-regulated: in androgen-responsive cell lines (LNCaP, VCaP), AR levels are higher in the presence of androgens (DHT, R1881). Include AR-positive (LNCaP) and AR-negative (PC-3 or DU145) cell line controls in each assay. For tissue samples, normalize AR concentration to total protein (BCA assay) and report as ng AR/mg total protein. The standard curve should be prepared in Standard Diluent; do not use Sample Diluent for standards (matrix effects). For detecting AR-V7 specifically in CTCs, consider using AR-V7-specific ELISA or digital droplet PCR for mRNA. |
| Batch-to-Batch Consistency | Standard curve R2 >0.99; LOD <0.05 ng/mL; AR recovery from LNCaP lysate within +/-20% of reference lot; inter-lot CV <15%. |
For research use only, not for clinical use.
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