Description
XIAP Colorimetric Cell-Based ELISA Kit
The XIAP (X-linked Inhibitor of Apoptosis Protein) Colorimetric Cell-Based ELISA Kit is specifically designed for the accurate and reliable detection of XIAP levels in cell lysates and culture supernatants. This kit offers high sensitivity and specificity, allowing for precise measurements and reproducible results in a variety of research applications.XIAP is a key regulator of apoptosis, playing a crucial role in cell survival and programmed cell death. Dysregulation of XIAP has been implicated in various diseases, including cancer, autoimmune disorders, and neurodegenerative diseases.
Therefore, measuring XIAP levels can provide valuable insights into disease mechanisms and potential therapeutic targets.With its user-friendly protocol and robust performance, the XIAP Colorimetric Cell-Based ELISA Kit is a valuable tool for researchers studying apoptosis, cell survival, and disease processes. Order yours today and unlock the potential of XIAP research in your laboratory.
| Product Name: | XIAP Colorimetric Cell-Based ELISA Kit |
| Product Code: | CBCAB00907 |
| ELISA Type: | Cell-Based |
| Target: | XIAP |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The XIAP Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect XIAP protein expression profile in cells. The kit can be used for measuring the relative amounts of XIAP in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on XIAP.
Qualitative determination of XIAP concentration is achieved by an indirect ELISA format. In essence, XIAP is captured by XIAP-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: 331, UniProt ID: P98170, OMIM: 300079/300635, Unigene: Hs.356076 |
| Gene Symbol: | XIAP |
| Sub Type: | None |
| UniProt Protein Function: | XIAP: an apoptotic suppressor protein. Inhibist apoptosis through binding to tumor necrosis factor receptor-associated factors TRAF1 and TRAF2. Inhibits apoptosis induced by menadione, a potent inducer of free radicals, and ICE. Inhibits cell-death proteases, caspase-3, caspase-7, and perhaps caspase-9. |
| UniProt Protein Details: | Protein type:EC 6.3.2.-; Apoptosis; Ubiquitin ligase; Ubiquitin conjugating system; Ligase Chromosomal Location of Human Ortholog: Xq25 Cellular Component: nucleoplasm; spindle microtubule; cytoplasm; nucleus; cytosol Molecular Function:protein binding; zinc ion binding; caspase inhibitor activity; ubiquitin-protein ligase activity; ligase activity Biological Process: Wnt receptor signaling pathway; positive regulation of protein ubiquitination; apoptosis; regulation of inflammatory response; regulation of BMP signaling pathway; protein ubiquitination; negative regulation of caspase activity; response to DNA damage stimulus; copper ion homeostasis; regulation of cell proliferation; negative regulation of apoptosis; regulation of innate immune response Disease: Lymphoproliferative Syndrome, X-linked, 1; Lymphoproliferative Syndrome, X-linked, 2 |
| NCBI Summary: | This gene encodes a protein that belongs to a family of apoptotic suppressor proteins. Members of this family share a conserved motif termed, baculovirus IAP repeat, which is necessary for their anti-apoptotic function. This protein functions through binding to tumor necrosis factor receptor-associated factors TRAF1 and TRAF2 and inhibits apoptosis induced by menadione, a potent inducer of free radicals, and interleukin 1-beta converting enzyme. This protein also inhibits at least two members of the caspase family of cell-death proteases, caspase-3 and caspase-7. Mutations in this gene are the cause of X-linked lymphoproliferative syndrome. Alternate splicing results in multiple transcript variants. Pseudogenes of this gene are found on chromosomes 2 and 11.[provided by RefSeq, Feb 2011] |
| UniProt Code: | P98170 |
| NCBI GenInfo Identifier: | 12643387 |
| NCBI Gene ID: | 331 |
| NCBI Accession: | P98170.2 |
| UniProt Secondary Accession: | P98170,Q9NQ14, D3DTF2, |
| UniProt Related Accession: | P98170 |
| Molecular Weight: | 497 |
| NCBI Full Name: | E3 ubiquitin-protein ligase XIAP |
| NCBI Synonym Full Names: | X-linked inhibitor of apoptosis, E3 ubiquitin protein ligase |
| NCBI Official Symbol: | XIAP |
| NCBI Official Synonym Symbols: | API3; ILP1; MIHA; XLP2; BIRC4; IAP-3; hIAP3; hIAP-3 |
| NCBI Protein Information: | E3 ubiquitin-protein ligase XIAP; X-linked IAP; IAP-like protein; inhibitor of apoptosis protein 3; baculoviral IAP repeat-containing protein 4 |
| UniProt Protein Name: | E3 ubiquitin-protein ligase XIAP |
| UniProt Synonym Protein Names: | Baculoviral IAP repeat-containing protein 4; IAP-like protein; ILP; hILP; Inhibitor of apoptosis protein 3; IAP-3; hIAP-3; hIAP3; X-linked inhibitor of apoptosis protein; X-linked IAP |
| Protein Family: | XIAP-associated factor |
| UniProt Gene Name: | XIAP |
| UniProt Entry Name: | XIAP_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-XIAP 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.)
