Ku80/XRCC5 (Phospho-Thr714) Colorimetric Cell-Based ELISA Kit
- SKU:
- CBCAB01281
- Product Type:
- ELISA Kit
- ELISA Type:
- Cell Based Phospho Specific
- Research Area:
- Epigenetics and Nuclear Signaling
- Reactivity:
- Human
- Detection Method:
- Colorimetric
Description
| Product Name: | Ku80/XRCC5 (Phospho-Thr714) Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB01281 |
| ELISA Type: | Cell-Based |
| Target: | Ku80/XRCC5 (Phospho-Thr714) |
| Reactivity: | Human |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nm |
| Format: | 2 x 96-Well Microplates |
The Ku80/XRCC5 (Phospho-Thr714) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Ku80/XRCC5 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Ku80/XRCC5 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on Ku80/XRCC5 phosphorylation.
Qualitative determination of Ku80/XRCC5 (Phospho-Thr714) concentration is achieved by an indirect ELISA format. In essence, Ku80/XRCC5 (Phospho-Thr714) is captured by Ku80/XRCC5 (Phospho-Thr714)-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: 7520, UniProt ID: P13010, OMIM: 194364, Unigene: Hs.388739 |
| Gene Symbol: | XRCC5 |
| Sub Type: | Phospho |
| UniProt Protein Function: | Ku80: the 80-kilodalton subunit of the Ku complex, also known as ATP-dependant DNA helicase II. A single stranded DNA-dependent ATP-dependent helicase. It functions together with the DNA ligase IV-XRCC4 complex in the repair of DNA double-strand break by non-homologous end joining and the completion of V(D)J recombination events. This gene functionally complements Chinese hamster xrs-6, a mutant defective in DNA double-strand break repair and in ability to undergo V(D)J recombination. A rare microsatellite polymorphism in this gene is associated with cancer in patients of varying radiosensitivity. Has a role in chromosome translocation. The DNA helicase II complex binds preferentially to fork-like ends of double-stranded DNA in a cell cycle-dependent manner. It works in the 3'-5' direction. Binding to DNA may be mediated by p70. |
| UniProt Protein Details: | Protein type:Nucleolus; RNA-binding; DNA-binding; EC 3.6.1.-; Nuclear receptor co-regulator; Helicase; EC 3.6.4.- Chromosomal Location of Human Ortholog: 2q35 Cellular Component: cytosol; membrane; nuclear chromosome, telomeric region; nuclear telomere cap complex; nucleolus; nucleoplasm; nucleus; plasma membrane Molecular Function:5'-deoxyribose-5-phosphate lyase activity; ATP binding; ATP-dependent DNA helicase activity; damaged DNA binding; DNA binding; double-stranded DNA binding; double-stranded telomeric DNA binding; protein binding; protein C-terminus binding; telomeric DNA binding; ubiquitin protein ligase binding Biological Process: cell proliferation; DNA duplex unwinding; DNA recombination; DNA repair; double-strand break repair; double-strand break repair via nonhomologous end joining; innate immune response; negative regulation of transcription, DNA-dependent; positive regulation of interferon type I production; positive regulation of neurogenesis; regulation of smooth muscle cell proliferation; telomere maintenance; transcription, DNA-dependent; viral reproduction |
| NCBI Summary: | The protein encoded by this gene is the 80-kilodalton subunit of the Ku heterodimer protein which is also known as ATP-dependant DNA helicase II or DNA repair protein XRCC5. Ku is the DNA-binding component of the DNA-dependent protein kinase, and it functions together with the DNA ligase IV-XRCC4 complex in the repair of DNA double-strand break by non-homologous end joining and the completion of V(D)J recombination events. This gene functionally complements Chinese hamster xrs-6, a mutant defective in DNA double-strand break repair and in ability to undergo V(D)J recombination. A rare microsatellite polymorphism in this gene is associated with cancer in patients of varying radiosensitivity. [provided by RefSeq, Jul 2008] |
| UniProt Code: | P13010 |
| NCBI GenInfo Identifier: | 125731 |
| NCBI Gene ID: | 7520 |
| NCBI Accession: | P13010.3 |
| UniProt Secondary Accession: | P13010,Q0Z7V0, Q4VBQ5, Q53HH7, Q7M4N0, Q9UCQ0, Q9UCQ1 A8K3X5, |
| UniProt Related Accession: | P13010 |
| Molecular Weight: | 82,705 Da |
| NCBI Full Name: | X-ray repair cross-complementing protein 5 |
| NCBI Synonym Full Names: | X-ray repair complementing defective repair in Chinese hamster cells 5 |
| NCBI Official Symbol: | XRCC5 |
| NCBI Official Synonym Symbols: | KU80; KUB2; Ku86; NFIV; KARP1; KARP-1 |
| NCBI Protein Information: | X-ray repair cross-complementing protein 5 |
| UniProt Protein Name: | X-ray repair cross-complementing protein 5 |
| UniProt Synonym Protein Names: | 86 kDa subunit of Ku antigen; ATP-dependent DNA helicase 2 subunit 2; ATP-dependent DNA helicase II 80 kDa subunit; CTC box-binding factor 85 kDa subunit; CTC85; CTCBF; DNA repair protein XRCC5; Ku80; Ku86; Lupus Ku autoantigen protein p86; Nuclear factor IV; Thyroid-lupus autoantigen; TLAA; X-ray repair complementing defective repair in Chinese hamster cells 5 (double-strand-break rejoining) |
| UniProt Gene Name: | XRCC5 |
| UniProt Entry Name: | XRCC5_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-Ku80/XRCC5 (Phospho-Thr714) Antibody, Anti-Ku80/XRCC5 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.)
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