Description
BRCA1 (Phospho-Ser1457)Colorimetric Cell-Based ELISA Kit
The BRCA1 Phospho-Ser1457 Colorimetric Cell-Based ELISA Kit is a cutting-edge assay designed for the precise detection of phosphorylated BRCA1 at serine 1457 in cell lysates. This kit is highly sensitive, offering reliable and reproducible results for a variety of research applications.BRCA1 is a critical tumor suppressor gene involved in DNA repair and maintenance of genomic stability. Phosphorylation of BRCA1 at serine 1457 has been linked to its function in DNA damage response and cell cycle regulation. Dysregulation of this phosphorylation event is associated with breast and ovarian cancer susceptibility, highlighting the importance of studying this marker in cancer research.
With the BRCA1 Phospho-Ser1457 Colorimetric Cell-Based ELISA Kit, researchers can accurately measure levels of phosphorylated BRCA1 in cell samples, providing valuable insights into cellular signaling pathways and potential therapeutic targets for cancer treatment. This kit is a valuable tool for advancing our understanding of BRCA1 biology and its role in cancer development.
| Product Name: | BRCA1 (Phospho-Ser1457)Colorimetric Cell-Based ELISA Kit |
| Product Code: | CBCAB01673 |
| ELISA Type: | Cell-Based |
| Target: | BRCA1 (Phospho-Ser1457) |
| Reactivity: | Human |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nm |
| Format: | 2 x 96-Well Microplates |
The BRCA1 (Phospho-Ser1457) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect BRCA1 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated BRCA1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on BRCA1 phosphorylation.
Qualitative determination of BRCA1 (Phospho-Ser1457) concentration is achieved by an indirect ELISA format. In essence, BRCA1 (Phospho-Ser1457) is captured by BRCA1 (Phospho-Ser1457)-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: 672, UniProt ID: P38398, OMIM: 113705/114480, Unigene: Hs.194143 |
| Gene Symbol: | BRCA1 |
| Sub Type: | Phospho |
| UniProt Protein Function: | E3 ubiquitin-protein ligase that specifically mediates the formation of 'Lys-6'-linked polyubiquitin chains and plays a central role in DNA repair by facilitating cellular responses to DNA damage. It is unclear whether it also mediates the formation of other types of polyubiquitin chains. The E3 ubiquitin-protein ligase activity is required for its tumor suppressor function. The BRCA1-BARD1 heterodimer coordinates a diverse range of cellular pathways such as DNA damage repair, ubiquitination and transcriptional regulation to maintain genomic stability. Regulates centrosomal microtubule nucleation. Required for normal cell cycle progression from G2 to mitosis. Required for appropriate cell cycle arrests after ionizing irradiation in both the S-phase and the G2 phase of the cell cycle. Involved in transcriptional regulation of P21 in response to DNA damage. Required for FANCD2 targeting to sites of DNA damage. May function as a transcriptional regulator. Inhibits lipid synthesis by binding to inactive phosphorylated ACACA and preventing its dephosphorylation. Contributes to homologous recombination repair (HRR) via its direct interaction with PALB2, fine-tunes recombinational repair partly through its modulatory role in the PALB2-dependent loading of BRCA2-RAD51 repair machinery at DNA breaks. Component of the BRCA1-RBBP8 complex which regulates CHEK1 activation and controls cell cycle G2/M checkpoints on DNA damage via BRCA1-mediated ubiquitination of RBBP8. Acts as a transcriptional activator (PubMed:20160719). |
| NCBI Summary: | This gene encodes a nuclear phosphoprotein that plays a role in maintaining genomic stability, and it also acts as a tumor suppressor. The encoded protein combines with other tumor suppressors, DNA damage sensors, and signal transducers to form a large multi-subunit protein complex known as the BRCA1-associated genome surveillance complex (BASC). This gene product associates with RNA polymerase II, and through the C-terminal domain, also interacts with histone deacetylase complexes. This protein thus plays a role in transcription, DNA repair of double-stranded breaks, and recombination. Mutations in this gene are responsible for approximately 40% of inherited breast cancers and more than 80% of inherited breast and ovarian cancers. Alternative splicing plays a role in modulating the subcellular localization and physiological function of this gene. Many alternatively spliced transcript variants, some of which are disease-associated mutations, have been described for this gene, but the full-length natures of only some of these variants has been described. A related pseudogene, which is also located on chromosome 17, has been identified. [provided by RefSeq, May 2009] |
| UniProt Code: | P38398 |
| NCBI GenInfo Identifier: | 728984 |
| NCBI Gene ID: | 672 |
| NCBI Accession: | P38398.2 |
| UniProt Secondary Accession: | P38398,O15129, Q1RMC1, Q3LRJ0, Q3LRJ6, Q6IN79, Q7KYU9 E9PFZ0, |
| UniProt Related Accession: | P38398 |
| Molecular Weight: | 208kDa |
| NCBI Full Name: | Breast cancer type 1 susceptibility protein |
| NCBI Synonym Full Names: | BRCA1, DNA repair associated |
| NCBI Official Symbol: | BRCA1 |
| NCBI Official Synonym Symbols: | IRIS; PSCP; BRCAI; BRCC1; FANCS; PNCA4; RNF53; BROVCA1; PPP1R53 |
| NCBI Protein Information: | breast cancer type 1 susceptibility protein |
| UniProt Protein Name: | Breast cancer type 1 susceptibility protein |
| UniProt Synonym Protein Names: | RING finger protein 53; RING-type E3 ubiquitin transferase BRCA1Curated |
| Protein Family: | BRCA1-A complex |
| UniProt Gene Name: | BRCA1 |
| 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-BRCA1 (Phospho-Ser1457) Antibody, Anti-BRCA1 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.)
