Description
TNFSF15 Colorimetric Cell-Based ELISA
The TNFSF15 Colorimetric Cell-Based ELISA Kit is specifically designed for the sensitive and accurate detection of TNFSF15 levels in cell lysates, serum, and plasma samples. This kit offers high sensitivity and specificity, providing reliable and reproducible results for researchers in various fields.TNFSF15, also known as TL1A, is a member of the tumor necrosis factor superfamily and plays a crucial role in regulating immune responses and inflammation.
Dysregulation of TNFSF15 has been linked to various autoimmune diseases, inflammatory conditions, and cancer, making it a valuable biomarker for studying these diseases and potential therapeutic interventions.With the TNFSF15 Colorimetric Cell-Based ELISA Kit, researchers can confidently measure TNFSF15 levels in their samples, advancing their understanding of the role of TNFSF15 in disease pathogenesis and progression.
| Product Name: | TNFSF15 Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB00960 |
| ELISA Type: | Cell-Based |
| Target: | TNFSF15 |
| Reactivity: | Human |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The TNFSF15 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect TNFSF15 protein expression profile in cells. The kit can be used for measuring the relative amounts of TNFSF15 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on TNFSF15.
Qualitative determination of TNFSF15 concentration is achieved by an indirect ELISA format. In essence, TNFSF15 is captured by TNFSF15-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: 9966, UniProt ID: O95150, OMIM: 604052, Unigene: Hs.241382 |
| Gene Symbol: | TNFSF15 |
| Sub Type: | None |
| UniProt Protein Function: | TNFSF15: Receptor for TNFRSF25 and TNFRSF6B. Mediates activation of NF-kappa-B. Inhibits vascular endothelial growth and angiogenesis (in vitro). Promotes activation of caspases and apoptosis. Belongs to the tumor necrosis factor family. 3 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Cytokine; Membrane protein, integral; Apoptosis Chromosomal Location of Human Ortholog: 9q32 Cellular Component: extracellular space; integral to plasma membrane; integral to membrane; plasma membrane Molecular Function:death receptor binding; cytokine activity; tumor necrosis factor receptor binding; receptor binding Biological Process: caspase activation; cytokine metabolic process; activation of NF-kappaB-inducing kinase; immune response; signal transduction; positive regulation of cytokine secretion |
| NCBI Summary: | The protein encoded by this gene is a cytokine that belongs to the tumor necrosis factor (TNF) ligand family. This protein is abundantly expressed in endothelial cells, but is not expressed in either B or T cells. The expression of this protein is inducible by TNF and IL-1 alpha. This cytokine is a ligand for receptor TNFRSF25 and decoy receptor TNFRSF21/DR6. It can activate NF-kappaB and MAP kinases, and acts as an autocrine factor to induce apoptosis in endothelial cells. This cytokine is also found to inhibit endothelial cell proliferation, and thus may function as an angiogenesis inhibitor. Two transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Feb 2011] |
| UniProt Code: | O95150 |
| NCBI GenInfo Identifier: | 189047102 |
| NCBI Gene ID: | 9966 |
| NCBI Accession: | O95150.2 |
| UniProt Secondary Accession: | O95150,Q3SX69, Q5VJK8, Q5VWH1, Q8NFE9, |
| UniProt Related Accession: | O95150 |
| Molecular Weight: | 20,131 Da |
| NCBI Full Name: | Tumor necrosis factor ligand superfamily member 15 |
| NCBI Synonym Full Names: | tumor necrosis factor (ligand) superfamily, member 15 |
| NCBI Official Symbol: | TNFSF15 |
| NCBI Official Synonym Symbols: | TL1; TL1A; VEGI; VEGI192A |
| NCBI Protein Information: | tumor necrosis factor ligand superfamily member 15; TNF superfamily ligand TL1A; TNF ligand-related molecule 1; vascular endothelial cell growth inhibitor; vascular endothelial growth inhibitor-192A |
| UniProt Protein Name: | Tumor necrosis factor ligand superfamily member 15 |
| UniProt Synonym Protein Names: | TNF ligand-related molecule 1; Vascular endothelial cell growth inhibitorCleaved into the following 2 chains:Tumor necrosis factor ligand superfamily member 15, membrane form; Tumor necrosis factor ligand superfamily member 15, secreted form |
| UniProt Gene Name: | TNFSF15 |
| UniProt Entry Name: | TNF15_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-TNFSF15 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.)
