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| Product Name | Estrogen Receptor Alpha (ER-alpha/ESR1) ELISA Kit, Quantitative, Cell and Tissue Lysate |
| Catalog No. | TMTR-HMM-0084 |
| Description | Sandwich ELISA kit for quantitative measurement of human estrogen receptor alpha (ER-alpha, ESR1) protein in cell and tissue lysates. ER-alpha is a ligand-activated nuclear transcription factor and the primary mediator of estrogen signaling in breast cancer, endometrial cancer, and ovarian cancer. ER-alpha expression status is the most important biomarker guiding endocrine therapy in breast cancer — ER-positive tumors respond to tamoxifen, aromatase inhibitors (letrozole, anastrozole, exemestane), and selective estrogen receptor degraders (fulvestrant, elacestrant). This ELISA provides a quantitative alternative to immunohistochemistry (IHC) for ER measurement, enabling precise quantitation for research studies on endocrine resistance, ER mutations (Y537S, D538G), and therapeutic response. The assay uses antibodies targeting distinct epitopes in the N-terminal activation function-1 (AF-1) and ligand-binding domains. |
| Intended Use | Quantitative ER-alpha protein measurement in: breast cancer cell lines (MCF-7, T-47D as ER+; MDA-MB-231, BT-549 as ER-); breast tumor tissue lysates; endometrial and ovarian cancer tissue lysates; xenograft tumor lysates; normal breast and uterine tissue; and studies of endocrine resistance mechanisms and ESR1 mutations. |
| Principle / Technology | Sandwich ELISA: anti-ER-alpha capture antibody (mouse monoclonal, AF-1 domain) binds ER-alpha; biotinylated anti-ER-alpha detection antibody (rabbit monoclonal, LBD) binds a second epitope; streptavidin-HRP/TMB colorimetric detection; quantitation against recombinant ER-alpha standard (full-length, insect cell-expressed). |
| Detection Method | Lyse cells/tissue in RIPA buffer with protease/phosphatase inhibitors; centrifuge; add 100 uL sample or standard; incubate 2 h RT; wash; add detection antibody 1 h; wash; streptavidin-HRP 30 min; wash; TMB 15-30 min; stop; read A450; quantitate from 4-PL standard curve. |
| Sample Type | Cell lysates (MCF-7, T-47D ER+, MDA-MB-231 ER-): 10-100 ug protein/well; breast tumor lysates: 20-200 ug/well; ER-positive cells: 10-50 ng ER/mg protein; ER-negative cells: <0.1 ng/mg. |
| Performance Range / Specifications | Standard curve: 0.078-5 ng/mL; LOD: <0.05 ng/mL; linear: 0.16-5 ng/mL R2 >0.99; detection in MCF-7: 10-50 ng/mg protein. |
| Sensitivity / LOD | LOD <0.05 ng/mL; ER detectable in approximately 5,000 MCF-7 cells; approximately 0.5 pM detection (66 kDa ER-alpha). |
| Specificity | Specific for human ER-alpha (ESR1); <0.1% cross-reactivity with ER-beta (ESR2), androgen receptor, progesterone receptor, glucocorticoid receptor at 10 ng/mL. Detects wild-type and common ESR1 mutants (Y537S, D538G). |
| Reaction Conditions / Protocol | Capture 2 h RT; detection 1 h RT; HRP 30 min RT; TMB 15-30 min; total ~4-5 h. |
| Components / Formulation | ER-alpha Antibody Coated 96-Well Plate, Recombinant Human ER-alpha Standard (lyophilized), Biotinylated Anti-ER-alpha Detection Antibody (100x), Streptavidin-HRP (100x), Sample Diluent, Standard Diluent, Wash Buffer (20x), TMB Substrate, Stop Solution, Plate Sealers, Protocol. |
| Storage Conditions | Plate and buffers at 2-8 C; Standard and antibodies at -20 C. |
| Shelf Life | 12 months from manufacture. |
| Package Specifications | 96 determinations. |
| Product Form | Pre-coated strip plate; liquid reagents; lyophilized standard. |
| Quality Control | Each lot: standard R2 >0.99; LOD <0.05 ng/mL; intra-CV <8%; inter-CV <12%; recovery 85-115% in MCF-7 lysate; no ER-beta cross-reactivity. |
| Key Features | Quantitative ER-alpha sandwich ELISA; detects wild-type and common mutants; validated in breast cancer cell lines; recombinant ER standard; 0.05 ng/mL sensitivity; strip plate format. |
| Purity | Antibodies >95%; recombinant ER standard >90% by SDS-PAGE; analytical grade reagents. |
| Concentration | Standard reconstituted at 5 ng/mL; antibodies 100x concentrate. |
| Activity / Unit Definition | Capture antibody Kd ~10^-10 M; detection antibody Kd ~10^-10 M. |
| Molecular Weight | ER-alpha: ~66 kDa (595 aa); insect cell-expressed standard includes post-translational modifications. |
| Source / Origin | Recombinant monoclonal antibodies (animal-free production); ER standard from baculovirus/insect cell expression; HRP plant-derived; animal-free except where noted. |
| pH Range / Optimal pH | Sample diluent pH 7.2-7.4; optimal binding pH 7.0-8.0. |
| Shipping Conditions | Cold pack; antibodies and standard on dry ice. |
| Expiration Date / Stability | 12 months; reconstituted standard 1 week at 2-8 C or 3 months at -80 C. |
| Regulatory / Compliance | For research use only; not for clinical diagnostic use. |
| Compatibility | RIPA lysis buffer compatible; SDS <0.05% final; DTT/2-ME up to 1 mM; protease/phosphatase inhibitors essential; normalize to total protein (BCA). |
| Recommended Buffer System | PBS pH 7.4, 1% BSA, 0.05% Tween-20. |
| Application Notes / Precautions | ER-alpha is regulated by estrogen: in MCF-7 cells, estradiol (E2) treatment downregulates ER protein over 24-48 h. For accurate basal ER measurement, culture cells in estrogen-depleted (charcoal-stripped) serum for 48-72 h before harvest. Include ER+ (MCF-7) and ER- (MDA-MB-231) controls. For tissue: snap-freeze immediately after surgical resection; avoid formalin-fixed samples (crosslinking alters epitopes). Normalize to total protein and report as ng ER/mg protein. For ESR1 mutation studies, this ELISA detects total ER protein but does not distinguish wild-type from mutant — combine with mutation-specific detection (ddPCR, NGS). |
| Batch-to-Batch Consistency | Standard R2 >0.99; LOD <0.05 ng/mL; MCF-7 recovery within +/-20%; inter-lot CV <15%. |
For research use only, not for clinical use.
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