Estrogen Receptor-beta (Phospho-Ser105) Colorimetric Cell-Based ELISA Kit
- SKU:
- CBCAB01424
- Product Type:
- ELISA Kit
- ELISA Type:
- Cell Based Phospho Specific
- Research Area:
- Epigenetics and Nuclear Signaling
- Reactivity:
- Human
- Reactivity:
- Mouse
- Reactivity:
- Rat
- Detection Method:
- Colorimetric
Description
| Product Name: | Estrogen Receptor-beta (Phospho-Ser105) Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB01424 |
| ELISA Type: | Cell-Based |
| Target: | Estrogen Receptor-beta (Phospho-Ser105) |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nm |
| Format: | 2 x 96-Well Microplates |
The Estrogen Receptor-beta (Phospho-Ser105) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Estrogen Receptor-beta protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Estrogen Receptor-beta in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on Estrogen Receptor-beta phosphorylation.
Qualitative determination of Estrogen Receptor-beta (Phospho-Ser105) concentration is achieved by an indirect ELISA format. In essence, Estrogen Receptor-beta (Phospho-Ser105) is captured by Estrogen Receptor-beta (Phospho-Ser105)-specific primary (1ø) antibodies while the HRP-conjugated secondary (2ø) antibodies bind the Fc region of the 1ø antibody. Through this binding, the HRP enzyme conjugated to the 2ø antibody can catalyze a colorimetric reaction upon substrate addition. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:
| 1. | A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values. |
| 2. | Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method may be used to determine cell density. After staining, the results can be analysed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted. |
| Database Information: | Gene ID: 2100, UniProt ID: Q92731, OMIM: 601663, Unigene: Hs.525392/Hs.660607 |
| Gene Symbol: | ESR2 |
| Sub Type: | Phospho |
| UniProt Protein Function: | ER-beta: a nuclear hormone receptor and transcription factor. Binds and activated by estrogen. Regulates gene expression and affects cellular proliferation and differentiation in target tissues. Binds estrogens with an affinity similar to that of ER-alpha, and activates expression of reporter genes containing estrogen response elements (ERE) in an estrogen-dependent manner. Eight alternatively-spliced isoforms have been described. Isoform beta-cx lacks ligand binding ability and has no or only very low ERE binding activity resulting in the loss of ligand-dependent transactivation ability. |
| UniProt Protein Details: | Protein type:DNA-binding; Nuclear receptor Chromosomal Location of Human Ortholog: 14q23.2 Cellular Component: nucleoplasm; mitochondrion; extracellular region; nucleus Molecular Function:receptor antagonist activity; protein binding; ligand-dependent nuclear receptor activity; estrogen receptor activity; enzyme binding; DNA binding; zinc ion binding; transcription coactivator activity; estrogen response element binding; steroid hormone receptor activity; transcription factor activity; steroid binding Biological Process: transcription initiation from RNA polymerase II promoter; estrogen receptor signaling pathway; induction of apoptosis by hormones; neuron migration; vagina development; negative regulation of transcription from RNA polymerase II promoter; uterus development; signal transduction; cell-cell signaling; ovarian follicle development; regulation of transcription, DNA-dependent; steroid hormone mediated signaling; gene expression; positive regulation of transcription factor activity; negative regulation of cell growth; brain development; negative regulation of epithelial cell proliferation |
| NCBI Summary: | This gene encodes a member of the family of estrogen receptors and superfamily of nuclear receptor transcription factors. The gene product contains an N-terminal DNA binding domain and C-terminal ligand binding domain and is localized to the nucleus, cytoplasm, and mitochondria. Upon binding to 17beta-estradiol or related ligands, the encoded protein forms homo- or hetero-dimers that interact with specific DNA sequences to activate transcription. Some isoforms dominantly inhibit the activity of other estrogen receptor family members. Several alternatively spliced transcript variants of this gene have been described, but the full-length nature of some of these variants has not been fully characterized. [provided by RefSeq, Jul 2008] |
| UniProt Code: | Q92731 |
| NCBI GenInfo Identifier: | 6166154 |
| NCBI Gene ID: | 2100 |
| NCBI Accession: | Q92731.2 |
| UniProt Related Accession: | Q92731 |
| Molecular Weight: | Predicted: 53 kDa |
| NCBI Full Name: | Estrogen receptor beta |
| NCBI Synonym Full Names: | estrogen receptor 2 (ER beta) |
| NCBI Official Symbol: | ESR2 |
| NCBI Official Synonym Symbols: | Erb; ESRB; ESTRB; NR3A2; ER-BETA; ESR-BETA |
| NCBI Protein Information: | estrogen receptor beta; estrogen receptor beta 4; nuclear receptor subfamily 3 group A member 2 |
| UniProt Protein Name: | 5p152 |
| UniProt Synonym Protein Names: | 5p152 |
| Protein Family: | Estrogen receptor |
| UniProt Gene Name: | ESR2 |
| UniProt Entry Name: | Q7LCB3_HUMAN |
| Component | Quantity |
| 96-Well Cell Culture Clear-Bottom Microplate | 2 plates |
| 10X TBS | 24 mL |
| Quenching Buffer | 24 mL |
| Blocking Buffer | 50 mL |
| 15X Wash Buffer | 50 mL |
| Primary Antibody Diluent | 12 mL |
| 100x Anti-Phospho Target Antibody | 60 µL |
| 100x Anti-Target Antibody | 60 µL |
| Anti-GAPDH Antibody | 60 µL |
| HRP-Conjugated Anti-Rabbit IgG Antibody | 12 mL |
| HRP-Conjugated Anti-Mouse IgG Antibody | 12 mL |
| SDS Solution | 12 mL |
| Stop Solution | 24 mL |
| Ready-to-Use Substrate | 12 mL |
| Crystal Violet Solution | 12 mL |
| Adhesive Plate Seals | 2 seals |
The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:
- Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional)
- Micropipettes with capability of measuring volumes ranging from 1 µL to 1 ml
- 37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
- Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
- Graph paper or computer software capable of generating or displaying logarithmic functions
- Absorbent papers or vacuum aspirator
- Test tubes or microfuge tubes capable of storing ≥1 ml
- Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
- Orbital shaker (optional)
- Deionized or sterile water
*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1. | Seed 200 µL of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µL of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37 °C prior to adding cells. |
| 2. | Incubate the cells for overnight at 37 °C, 5% CO2. |
| 3. | Treat the cells as desired. |
| 4. | Remove the cell culture medium and rinse with 200 µL of 1x TBS, twice. |
| 5. | Fix the cells by incubating with 100 µL of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells. |
| 6. | Remove the Fixing Solution and wash the plate 3 times with 200 µL 1x Wash Buffer for five minutes each time with gentle shaking on the orbital shaker. The plate can be stored at 4 °C for a week. |
| 7. | Add 100 µL of Quenching Buffer and incubate for 20 minutes at room temperature. |
| 8. | Wash the plate 3 times with 1x Wash Buffer for 5 minutes each time. |
| 9. | Add 200 µL of Blocking Buffer and incubate for 1 hour at room temperature. |
| 10. | Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time. |
| 11. | Add 50 µL of 1x primary antibodies Anti-Estrogen Receptor-beta (Phospho-Ser105) Antibody, Anti-Estrogen Receptor-beta Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover with Parafilm and incubate for 16 hours (overnight) at 4 °C. If the target expression is known to be high, incubate for 2 hours at room temperature. |
| 12. | Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time. |
| 13. | Add 50 µL of 1x secondary antibodies (HRP-Conjugated AntiRabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature. |
| 14. | Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time. |
| 15. | Add 50 µL of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark. |
| 16. | Add 50 µL of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader. |
(Additional Crystal Violet staining may be performed if desired – details of this may be found in the kit technical manual.)
Related Products
