Description
TRIP13 Colorimetric Cell-Based ELISA
The TRIP13 Colorimetric Cell-Based ELISA Kit is a cutting-edge assay designed for the sensitive and specific detection of TRIP13 levels in cell lysates and tissue homogenates. This kit offers high accuracy and precision, providing researchers with reliable and reproducible results for their studies.TRIP13 is a key protein involved in cell division and DNA repair processes, making it a vital target for cancer research and drug development.
By measuring TRIP13 levels, researchers can gain valuable insights into the mechanisms underlying tumorigenesis and potentially identify new therapeutic strategies for cancer treatment.Overall, the TRIP13 Colorimetric Cell-Based ELISA Kit is an invaluable tool for studying TRIP13 function and its significance in various cellular processes, offering researchers a powerful platform for advancing their scientific discoveries.
| Product Name: | TRIP13 Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB01111 |
| ELISA Type: | Cell-Based |
| Target: | TRIP13 |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The TRIP13 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect TRIP13 protein expression profile in cells. The kit can be used for measuring the relative amounts of TRIP13 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on TRIP13.
Qualitative determination of TRIP13 concentration is achieved by an indirect ELISA format. In essence, TRIP13 is captured by TRIP13-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: 9319, UniProt ID: Q15645, OMIM: 604507, Unigene: Hs.436187 |
| Gene Symbol: | TRIP13 |
| Sub Type: | None |
| UniProt Protein Function: | TRIP13: Plays a key role in chromosome recombination and chromosome structure development during meiosis. Required at early steps in meiotic recombination that leads to non-crossovers pathways. Also needed for efficient completion of homologous synapsis by influencing crossover distribution along the chromosomes affecting both crossovers and non-crossovers pathways. Also required for development of higher-order chromosome structures and is needed for synaptonemal-complex formation. In males, required for efficient synapsis of the sex chromosomes and for sex body formation. Promotes early steps of the DNA double- strand breaks (DSBs) repair process upstream of the assembly of RAD51 complexes. Required for depletion of HORMAD1 and HORMAD2 from synapsed chromosomes. Belongs to the AAA ATPase family. PCH2 subfamily. 2 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Nuclear receptor co-regulator; Transcription, coactivator/corepressor Chromosomal Location of Human Ortholog: 5p15.33 Cellular Component: nucleus Molecular Function:identical protein binding; protein binding; transcription cofactor activity Biological Process: double-strand break repair; meiotic recombination; oogenesis; spermatogenesis; synaptonemal complex assembly; transcription from RNA polymerase II promoter |
| NCBI Summary: | This gene encodes a protein that interacts with thyroid hormone receptors, also known as hormone-dependent transcription factors. The gene product interacts specifically with the ligand binding domain. This gene is one of several that may play a role in early-stage non-small cell lung cancer. [provided by RefSeq, Oct 2009] |
| UniProt Code: | Q15645 |
| NCBI GenInfo Identifier: | 85541056 |
| NCBI Gene ID: | 9319 |
| NCBI Accession: | Q15645.2 |
| UniProt Secondary Accession: | Q15645,O15324, C9K0T3, D3DTC0, |
| UniProt Related Accession: | Q15645 |
| Molecular Weight: | 22,084 Da |
| NCBI Full Name: | Pachytene checkpoint protein 2 homolog |
| NCBI Synonym Full Names: | thyroid hormone receptor interactor 13 |
| NCBI Official Symbol: | TRIP13 |
| NCBI Official Synonym Symbols: | 16E1BP |
| NCBI Protein Information: | pachytene checkpoint protein 2 homolog |
| UniProt Protein Name: | Pachytene checkpoint protein 2 homolog |
| UniProt Synonym Protein Names: | Human papillomavirus type 16 E1 protein-binding protein; 16E1-BP; HPV16 E1 protein-binding protein; Thyroid hormone receptor interactor 13; Thyroid receptor-interacting protein 13; TR-interacting protein 13; TRIP-13 |
| UniProt Gene Name: | TRIP13 |
| UniProt Entry Name: | PCH2_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-TRIP13 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.)
