Description
ER81 Colorimetric Cell-Based ELISA
The ER81 Colorimetric Cell-Based ELISA Kit is a cutting-edge assay designed for the detection of ER81 levels in cell culture samples. This kit offers high sensitivity and specificity, allowing for accurate and reproducible results for research applications.ER81 is a transcription factor that plays a critical role in cell growth and differentiation, making it a valuable biomarker for studying various cellular processes and diseases.
By measuring ER81 levels in cell culture supernatants, researchers can gain insights into the molecular mechanisms underlying different cellular pathways and diseases.With its easy-to-use protocol and reliable performance, the ER81 Colorimetric Cell-Based ELISA Kit is an essential tool for researchers looking to explore the role of ER81 in cellular biology and disease pathogenesis.
| Product Name: | ER81 Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB01024 |
| ELISA Type: | Cell-Based |
| Target: | ER81 |
| Reactivity: | Human, Mouse |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The ER81 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect ER81 protein expression profile in cells. The kit can be used for measuring the relative amounts of ER81 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on ER81.
Qualitative determination of ER81 concentration is achieved by an indirect ELISA format. In essence, ER81 is captured by ER81-specific primary antibodies while the HRP-conjugated secondary antibodies bind the Fc region of the primary antibody. Through this binding, the HRP enzyme conjugated to the secondary 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: 2115, UniProt ID: P50549, OMIM: 600541, Unigene: Hs.22634 |
| Gene Symbol: | ETV1 |
| Sub Type: | None |
| UniProt Protein Function: | ETV1: Transcriptional activator that binds to DNA sequences containing the consensus pentanucleotide 5'-CGGA[AT]-3'. Defects in ETV1 are a cause of Ewing sarcoma (ES). A highly malignant, metastatic, primitive small round cell tumor of bone and soft tissue that affects children and adolescents. It belongs to the Ewing sarcoma family of tumors, a group of morphologically heterogeneous neoplasms that share the same cytogenetic features. They are considered neural tumors derived from cells of the neural crest. Ewing sarcoma represents the less differentiated form of the tumors. A chromosomal aberration involving ETV1 is found in patients with Erwing sarcoma. Translocation t(7;22)(p22;q12) with EWSR1. Belongs to the ETS family. 4 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Oncoprotein; Transcription factor; DNA-binding Chromosomal Location of Human Ortholog: 7p21.3 Cellular Component: nucleus Molecular Function:protein binding; transcription factor activity Biological Process: peripheral nervous system neuron development; positive regulation of transcription from RNA polymerase II promoter |
| NCBI Summary: | This gene encodes a member of the ETS (E twenty-six) family of transcription factors. The ETS proteins regulate many target genes that modulate biological processes like cell growth, angiogenesis, migration, proliferation and differentiation. All ETS proteins contain an ETS DNA-binding domain that binds to DNA sequences containing the consensus 5'-CGGA[AT]-3'. The protein encoded by this gene contains a conserved short acidic transactivation domain (TAD) in the N-terminal region, in addition to the ETS DNA-binding domain in the C-terminal region. This gene is involved in chromosomal translocations, which result in multiple fusion proteins including EWS-ETV1 in Ewing sarcoma and at least 10 ETV1 partners (see PMID: 19657377, Table 1) in prostate cancer. In addition to chromosomal rearrangement, this gene is overexpressed in prostate cancer, melanoma and gastrointestinal stromal tumor. Multiple alternatively spliced transcript variants encoding different isoforms have been identified. [provided by RefSeq, Jul 2016] |
| UniProt Code: | P50549 |
| NCBI GenInfo Identifier: | 12643411 |
| NCBI Gene ID: | 2115 |
| NCBI Accession: | P50549.2 |
| UniProt Secondary Accession: | P50549,O75849, Q4KMQ6, Q59GA7, A4D118, B2R768, B7Z2I4 B7Z618, B7Z9P2, C9JT37, E9PHB1, F5GXR2, |
| UniProt Related Accession: | P50549 |
| Molecular Weight: | 50,217 Da |
| NCBI Full Name: | ETS translocation variant 1 |
| NCBI Synonym Full Names: | ETS variant 1 |
| NCBI Official Symbol: | ETV1 |
| NCBI Official Synonym Symbols: | ER81 |
| NCBI Protein Information: | ETS translocation variant 1 |
| UniProt Protein Name: | ETS translocation variant 1 |
| UniProt Synonym Protein Names: | Ets-related protein 81 |
| Protein Family: | ETS translocation |
| UniProt Gene Name: | ETV1 |
| UniProt Entry Name: | ETV1_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-ER81 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.)
