Description
Human FAS/CD95 (Factor Related Apoptosis) ELISA Kit
The Human FAS (CD95) Factor-Related Apoptosis ELISA Kit is designed for the accurate detection of FAS levels in human serum, plasma, and cell culture supernatants. This kit features high sensitivity and specificity, ensuring reliable and reproducible results, making it ideal for a wide range of research applications.FAS, also known as CD95, is a cell surface receptor involved in apoptosis, or programmed cell death. Dysregulation of the FAS pathway has been implicated in various diseases, including cancer and autoimmune disorders, making it a valuable biomarker for studying these conditions and developing potential therapies.
With the Human FAS Factor-Related Apoptosis ELISA Kit, researchers can accurately measure FAS levels in biological samples, providing valuable insights into cell death pathways and potential therapeutic targets. Trust Assay Genie for high-quality ELISA kits for your research needs.
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Human |
| Detection Method: | Colormetric |
| Detection Range: | 31.25-2000 pg/mL |
| Sensitivity: | 18.75 pg/mL |
| Sample Volume Required Per Well: | 100µL |
| Sample Type: | Serum, plasma and other biological fluids |
| Specificity: | This kit recognizes Human FAS/CD95 in samples. No significant cross-reactivity or interference between Human FAS/CD95 and analogues was observed. |
This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Human FAS/CD95. Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human FAS/CD95 and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Human FAS/CD95, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Human FAS/CD95. The concentration of Human FAS/CD95 in samples can be calculated by comparing the OD of the samples to the standard curve.
| UniProt Protein Function: | FAS: Receptor for TNFSF6/FASLG. The adapter molecule FADD recruits caspase-8 to the activated receptor. The resulting death- inducing signaling complex (DISC) performs caspase-8 proteolytic activation which initiates the subsequent cascade of caspases (aspartate-specific cysteine proteases) mediating apoptosis. FAS- mediated apoptosis may have a role in the induction of peripheral tolerance, in the antigen-stimulated suicide of mature T-cells, or both. The secreted isoforms 2 to 6 block apoptosis (in vitro). Binds DAXX. Interacts with HIPK3. Part of a complex containing HIPK3 and FADD. Binds RIPK1 and FAIM2. Interacts with BRE and FEM1B. Interacts with FADD. Isoform 1 and isoform 6 are expressed at equal levels in resting peripheral blood mononuclear cells. After activation there is an increase in isoform 1 and decrease in the levels of isoform 6. 6 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Apoptosis; Membrane protein, integral; Receptor, cytokine; Cell surface Chromosomal Location of Human Ortholog: 10q24. 1 Cellular Component: cell surface; cytoplasm; plasma membrane; integral to membrane; CD95 death-inducing signaling complex; nucleus; cytosol; lipid raft; external side of plasma membrane Molecular Function:identical protein binding; protein binding; signal transducer activity; transmembrane receptor activity; receptor activity; kinase binding Biological Process: circadian rhythm; caspase activation; spleen development; regulation of myeloid cell differentiation; renal system process; transformed cell apoptosis; apoptosis; positive regulation of apoptosis; positive regulation of protein homooligomerization; regulation of lymphocyte differentiation; response to toxin; response to glucocorticoid stimulus; negative regulation of B cell activation; signal transduction; negative thymic T cell selection; regulation of apoptosis; inflammatory cell apoptosis; B cell mediated immunity; induction of apoptosis via death domain receptors; gene expression; protein complex assembly; immunoglobulin production; activated T cell apoptosis; protein homooligomerization; negative regulation of apoptosis Disease: Autoimmune Lymphoproliferative Syndrome |
| NCBI Summary: | The protein encoded by this gene is a member of the TNF-receptor superfamily. This receptor contains a death domain. It has been shown to play a central role in the physiological regulation of programmed cell death, and has been implicated in the pathogenesis of various malignancies and diseases of the immune system. The interaction of this receptor with its ligand allows the formation of a death-inducing signaling complex that includes Fas-associated death domain protein (FADD), caspase 8, and caspase 10. The autoproteolytic processing of the caspases in the complex triggers a downstream caspase cascade, and leads to apoptosis. This receptor has been also shown to activate NF-kappaB, MAPK3/ERK1, and MAPK8/JNK, and is found to be involved in transducing the proliferating signals in normal diploid fibroblast and T cells. Several alternatively spliced transcript variants have been described, some of which are candidates for nonsense-mediated mRNA decay (NMD). The isoforms lacking the transmembrane domain may negatively regulate the apoptosis mediated by the full length isoform. [provided by RefSeq, Mar 2011] |
| UniProt Code: | P25445 |
| NCBI GenInfo Identifier: | 119833 |
| NCBI Gene ID: | 355 |
| NCBI Accession: | P25445. 1 |
| UniProt Secondary Accession: | P25445,Q14292, Q14293, Q14294, Q14295, Q16652, Q5T9P1 Q5T9P2, Q5T9P3, Q6SSE9, A9UJX4, B6VNV4, |
| UniProt Related Accession: | P25445 |
| Molecular Weight: | 37,732 Da |
| NCBI Full Name: | Tumor necrosis factor receptor superfamily member 6 |
| NCBI Synonym Full Names: | Fas cell surface death receptor |
| NCBI Official Symbol: | FAS |
| NCBI Official Synonym Symbols: | APT1; CD95; FAS1; APO-1; FASTM; ALPS1A; TNFRSF6 |
| NCBI Protein Information: | tumor necrosis factor receptor superfamily member 6; Fas AMA; FAS 827dupA; CD95 antigen; FASLG receptor; apoptosis antigen 1; Delta Fas/APO-1/CD95; FAS receptor variant 9; APO-1 cell surface antigen; TNF receptor superfamily member 6; apoptosis-mediating surface antigen FAS; Fas (TNF receptor superfamily, member 6); tumor necrosis factor receptor superfamily, member 6 |
| UniProt Protein Name: | Tumor necrosis factor receptor superfamily member 6 |
| UniProt Synonym Protein Names: | Apo-1 antigen; Apoptosis-mediating surface antigen FAS; FASLG receptor |
| Protein Family: | Development-specific protein |
| UniProt Gene Name: | FAS |
| UniProt Entry Name: | TNR6_HUMAN |
As the OD values of the standard curve may vary according to the conditions of the actual assay performance (e. g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.
| Concentration (pg/mL) | O.D | Average | Corrected |
| 2000 | 2.342 2.356 | 2.349 | 2.291 |
| 1000 | 1.611 1.633 | 1.622 | 1.564 |
| 500 | 0.97 0.962 | 0.966 | 0.908 |
| 250 | 0.503 0.509 | 0.506 | 0.448 |
| 125 | 0.252 0.226 | 0.239 | 0.181 |
| 62.5 | 0.166 0.152 | 0.159 | 0.101 |
| 31.25 | 0.105 0.115 | 0.11 | 0.052 |
| 0 | 0.052 0.064 | 0.058 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human FAS/CD95 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human FAS/CD95 were tested on 3 different plates, 20 replicates in each plate.
| Intra-assay Precision | Inter-assay Precision | |||||
| Sample | 1 | 2 | 3 | 1 | 2 | 3 |
| n | 20 | 20 | 20 | 20 | 20 | 20 |
| Mean (pg/mL) | 96.87 | 296.85 | 832.96 | 104.02 | 325.88 | 858.57 |
| Standard deviation | 5.68 | 16.42 | 43.65 | 6.22 | 14.44 | 29.79 |
| C V (%) | 5.86 | 5.53 | 5.24 | 5.98 | 4.43 | 3.47 |
Recovery
The recovery of Human FAS/CD95 spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.
| Sample Type | Range (%) | Average Recovery (%) |
| Serum (n=5) | 93-107 | 99 |
| EDTA plasma (n=5) | 87-98 | 93 |
| Cell culture media (n=5) | 85-96 | 91 |
Linearity
Samples were spiked with high concentrations of Human FAS/CD95 and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.
| Serum (n=5) | EDTA plasma (n=5) | Cell culture media (n=5) | ||
| 1:2 | Range (%) | 94-106 | 93-110 | 92-105 |
| Average (%) | 100 | 100 | 98 | |
| 1:4 | Range (%) | 93-105 | 78-92 | 84-94 |
| Average (%) | 99 | 85 | 89 | |
| 1:8 | Range (%) | 91-104 | 80-94 | 82-92 |
| Average (%) | 96 | 86 | 87 | |
| 1:16 | Range (%) | 87-99 | 79-90 | 87-100 |
| Average (%) | 93 | 86 | 94 |
An unopened kit can be stored at 4°C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.
| Item | Specifications | Storage |
| Micro ELISA Plate(Dismountable) | 8 wells ×12 strips | -20°C, 6 months |
| Reference Standard | 2 vials | |
| Concentrated Biotinylated Detection Ab (100×) | 1 vial, 120 µL | |
| Concentrated HRP Conjugate (100×) | 1 vial, 120 µL | -20°C(shading light), 6 months |
| Reference Standard & Sample Diluent | 1 vial, 20 mL | 4°C, 6 months |
| Biotinylated Detection Ab Diluent | 1 vial, 14 mL | |
| HRP Conjugate Diluent | 1 vial, 14 mL | |
| Concentrated Wash Buffer (25×) | 1 vial, 30 mL | |
| Substrate Reagent | 1 vial, 10 mL | 4°C(shading light) |
| Stop Solution | 1 vial, 10 mL | 4°C |
| Plate Sealer | 5 pieces | |
| Product Description | 1 copy | |
| Certificate of Analysis | 1 copy |
- Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
- Aliquot 100 µL of standard solutions into the standard wells.
- Add 100 µL of Sample / Standard dilution buffer into the control (zero) well.
- Add 100 µL of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
- Cover the plate with the sealer provided in the kit and incubate for 90 min at 37 °C.
- Aspirate the liquid from each well, do not wash. Immediately add 100 µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix. Incubate for 1 hour at 37 °C.
- Aspirate or decant the solution from the plate and add 350 µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
- Add 100 µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37 °C.
- Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
- Add 90 µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37 °C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
- Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
- Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.
