Description
RAD51 (Phospho-Tyr315)Colorimetric Cell-Based ELISA Kit
The RAD51 Phospho-Tyr315 Colorimetric Cell-Based ELISA Kit is specifically designed for the accurate detection of phosphorylated RAD51 at tyrosine 315 in cell lysates. This kit offers high sensitivity and specificity, providing reliable and reproducible results for a variety of research applications.RAD51 is a key protein involved in DNA repair and homologous recombination, playing a critical role in maintaining genomic stability. Phosphorylation of RAD51 at tyrosine 315 has been linked to DNA damage response and repair processes, making it a valuable biomarker for studying cellular responses to genotoxic stress and potential therapeutic targets for cancer and other diseases.
With easy-to-follow protocols and user-friendly instructions, the RAD51 Phospho-Tyr315 Colorimetric Cell-Based ELISA Kit is a powerful tool for researchers looking to study RAD51 phosphorylation dynamics and its impact on DNA repair mechanisms.
| Product Name: | RAD51 (Phospho-Tyr315)Colorimetric Cell-Based ELISA Kit |
| Product Code: | CBCAB01463 |
| ELISA Type: | Cell-Based |
| Target: | RAD51 (Phospho-Tyr315) |
| Reactivity: | Human, Mouse |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nm |
| Format: | 2 x 96-Well Microplates |
The RAD51 (Phospho-Tyr315) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect RAD51 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated RAD51 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on RAD51 phosphorylation.
Qualitative determination of RAD51 (Phospho-Tyr315) concentration is achieved by an indirect ELISA format. In essence, RAD51 (Phospho-Tyr315) is captured by RAD51 (Phospho-Tyr315)-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: 5888, UniProt ID: Q06609, OMIM: 114480/179617, Unigene: Hs.631709 |
| Gene Symbol: | RAD51 |
| Sub Type: | Phospho |
| UniProt Protein Function: | Plays an important role in homologous strand exchange, a key step in DNA repair through homologous recombination (HR). Binds to single and double-stranded DNA and exhibits DNA-dependent ATPase activity. Catalyzes the recognition of homology and strand exchange between homologous DNA partners to form a joint molecule between a processed DNA break and the repair template. Binds to single-stranded DNA in an ATP-dependent manner to form nucleoprotein filaments which are essential for the homology search and strand exchange (PubMed:26681308). Part of a PALB2-scaffolded HR complex containing BRCA2 and RAD51C and which is thought to play a role in DNA repair by HR. Plays a role in regulating mitochondrial DNA copy number under conditions of oxidative stress in the presence of RAD51C and XRCC3. Also involved in interstrand cross-link repair (PubMed:26253028). |
| NCBI Summary: | The protein encoded by this gene is a member of the RAD51 protein family. RAD51 family members are highly similar to bacterial RecA and Saccharomyces cerevisiae Rad51, and are known to be involved in the homologous recombination and repair of DNA. This protein can interact with the ssDNA-binding protein RPA and RAD52, and it is thought to play roles in homologous pairing and strand transfer of DNA. This protein is also found to interact with BRCA1 and BRCA2, which may be important for the cellular response to DNA damage. BRCA2 is shown to regulate both the intracellular localization and DNA-binding ability of this protein. Loss of these controls following BRCA2 inactivation may be a key event leading to genomic instability and tumorigenesis. Multiple transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Aug 2009] |
| UniProt Code: | Q06609 |
| NCBI GenInfo Identifier: | 548663 |
| NCBI Gene ID: | 5888 |
| NCBI Accession: | Q06609.1 |
| UniProt Secondary Accession: | Q06609,Q6FHX9, Q6ZNA8, Q9BV60, B0FXP0, B2R8T6, |
| UniProt Related Accession: | Q06609 |
| Molecular Weight: | |
| NCBI Full Name: | DNA repair protein RAD51 homolog 1 |
| NCBI Synonym Full Names: | RAD51 recombinase |
| NCBI Official Symbol: | RAD51 |
| NCBI Official Synonym Symbols: | RECA; BRCC5; FANCR; MRMV2; HRAD51; RAD51A; HsRad51; HsT16930 |
| NCBI Protein Information: | DNA repair protein RAD51 homolog 1 |
| UniProt Protein Name: | DNA repair protein RAD51 homolog 1 |
| UniProt Synonym Protein Names: | RAD51 homolog A |
| Protein Family: | DNA repair protein |
| UniProt Gene Name: | RAD51 |
| UniProt Entry Name: | RAD51_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-RAD51 (Phospho-Tyr315) Antibody, Anti-RAD51 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.)
