Description
APAF-1-ALT Colorimetric Cell-Based ELISA Kit
The APAF-1 (ALT) Colorimetric Cell-Based ELISA Kit is a reliable and accurate tool for detecting APAF-1 (ALT) levels in cell lysates and tissue homogenates. This kit offers high sensitivity and specificity, providing consistent and reproducible results for a variety of experimental applications.APAF-1 (ALT) is a key protein involved in the apoptotic pathway, playing a crucial role in cell death regulation and immune response. Dysregulation of APAF-1 (ALT) has been implicated in various diseases, including cancer and autoimmune disorders, making it a valuable biomarker for studying these conditions and developing targeted therapies.
With its user-friendly protocol and rapid assay time, the APAF-1 (ALT) Colorimetric Cell-Based ELISA Kit is a valuable addition to any research laboratory looking to investigate the role of APAF-1 (ALT) in disease pathology and therapeutic interventions.
| Product Name: | APAF-1-ALT Colorimetric Cell-Based ELISA Kit |
| Product Code: | CBCAB00529 |
| ELISA Type: | Cell-Based |
| Target: | APAF-1-ALT |
| Reactivity: | Human |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The APAF-1-ALT Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect APAF-1-ALT protein expression profile in cells. The kit can be used for measuring the relative amounts of APAF-1-ALT in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on APAF-1-ALT.
Qualitative determination of APAF-1-ALT concentration is achieved by an indirect ELISA format. In essence, APAF-1-ALT is captured by APAF-1-ALT-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: 317, UniProt ID: O14727-6, OMIM: 602233, Unigene: Hs.724521 |
| Gene Symbol: | APAF1 |
| Sub Type: | None |
| UniProt Protein Function: | APAF: Oligomeric Apaf-1 mediates the cytochrome c-dependent autocatalytic activation of pro-caspase-9 (Apaf-3), leading to the activation of caspase-3 and apoptosis. This activation requires ATP. Isoform 6 is less effective in inducing apoptosis. Monomer. Oligomerizes upon binding of cytochrome c and dATP. Oligomeric Apaf-1 and pro-caspase-9 bind to each other via their respective NH2-terminal CARD domains and consecutively mature caspase-9 is released from the complex. Pro-caspase-3 is recruited into the Apaf-1-pro-caspase-9 complex via interaction with pro-caspase-9. Interacts with APIP. Interacts (via CARD and NACHT domains) with NAIP/BIRC1 (via NACHT domain). By E2F and p53/TP53 in apoptotic neurons. Ubiquitous. Highest levels of expression in adult spleen and peripheral blood leukocytes, and in fetal brain, kidney and lung. Isoform 1 is expressed in heart, kidney and liver. 6 isoforms of the human protein are produced by alternative splicing. Protein type: Apoptosis Chromosomal Location of Human Ortholog: 12q23.1 Cellular Component: apoptosome; cytosol Molecular Function: nucleotide binding; protein binding Biological Process: apoptosis; caspase activation; caspase activation via cytochrome c; nervous system development; positive regulation of apoptosis; regulation of apoptosis |
| UniProt Protein Details: | |
| NCBI Summary: | This gene encodes a cytoplasmic protein that initiates apoptosis. This protein contains several copies of the WD-40 domain, a caspase recruitment domain (CARD), and an ATPase domain (NB-ARC). Upon binding cytochrome c and dATP, this protein forms an oligomeric apoptosome. The apoptosome binds and cleaves caspase 9 preproprotein, releasing its mature, activated form. Activated caspase 9 stimulates the subsequent caspase cascade that commits the cell to apoptosis. Alternative splicing results in several transcript variants encoding different isoforms. [provided by RefSeq, Jul 2008] |
| UniProt Code: | O14727-6 |
| NCBI GenInfo Identifier: | 20141188 |
| NCBI Gene ID: | 317 |
| NCBI Accession: | O14727.2 |
| UniProt Secondary Accession: | O14727-6,O43297, Q7Z438, Q9BXZ6, Q9UBZ5, Q9UGN8, Q9UGN9 Q9UGP0, Q9UJ58, Q9UJ59 |
| UniProt Related Accession: | O14727-6,O14727 |
| Molecular Weight: | 37,976 Da |
| NCBI Full Name: | Apoptotic protease-activating factor 1 |
| NCBI Synonym Full Names: | apoptotic peptidase activating factor 1 |
| NCBI Official Symbol: | APAF1 |
| NCBI Official Synonym Symbols: | CED4; APAF-1 |
| NCBI Protein Information: | apoptotic protease-activating factor 1 |
| UniProt Protein Name: | Apoptotic protease-activating factor 1 |
| UniProt Synonym Protein Names: | |
| Protein Family: | Apoptotic protease-activating factor |
| UniProt Gene Name: | APAF1 |
| UniProt Entry Name: |
| 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-APAF-1-ALT 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.)
