Human APAF1 (Apoptosis Protease Activating Factor 1) ELISA Kit
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 APAF1 . 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 APAF1 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 APAF1, 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 APAF1. The concentration of Human APAF1 in samples can be calculated by comparing the OD of the samples to the standard curve.
|Detection Range||0.16-10 ng/mL|
|Sample Volume Required Per Well||100uL|
|Sample Type||Serum, plasma and other biological fluids|
This kit recognizes Human APAF1 in samples. No significant cross-reactivity or interference between Human APAF1 and analogues was observed.
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.
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human APAF1 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human APAF1 were tested on 3 different plates, 20 replicates in each plate.
|Intra-assay Precision||Inter-assay Precision|
|C V (%)||4.35||4.84||3.35||7.14||6.03||4.47|
The recovery of Human APAF1 spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.
|Sample Type||Range (%)||Average Recovery (%)|
|EDTA plasma (n=5)||92-108||100|
|Cell culture media (n=5)||84-99||91|
Samples were spiked with high concentrations of Human APAF1 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)|
Kit Components & Storage
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.
|Micro ELISA Plate(Dismountable)||8 wells X12 strips||-20'C, 6 months|
|Reference Standard||2 vials|
|Concentrated Biotinylated Detection Ab (100X)||1 vial, 120 uL|
|Concentrated HRP Conjugate (100X)||1 vial, 120 uL||-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 (25X)||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|
Human APAF1 (Apoptosis Protease Activating Factor 1) ELISA Kit (HUES01685) Assay procedure
- 1. 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.
- 2. Aliquot 100 µL of standard solutions into the standard wells.
- 3. Add 100 µL of Sample / Standard dilution buffer into the control (zero) well.
- 4. Add 100 µL of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
- 5. Cover the plate with the sealer provided in the kit and incubate for 90 min at 37 °C.
- 6. 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.
- 7. 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.
- 8. Add 100 µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37 °C.
- 9. Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
- 10. 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.
- 11. Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
- 12. Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.
Human APAF1 (Apoptosis Protease Activating Factor 1) ELISA Kit (HUES01685) Protein Information
|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.|
|UniProt Protein Details:|
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
|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]|
|NCBI GenInfo Identifier:||20141188|
|NCBI Gene ID:||317|
|UniProt Secondary Accession:||O14727,O43297, Q7Z438, Q9BXZ6, Q9UBZ5, Q9UGN8, Q9UGN9 Q9UGP0, Q9UJ58, Q9UJ59, Q9UJ60, B2RMX8,|
|UniProt Related Accession:||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|
|Protein Family:||Apoptotic protease-activating factor|
|UniProt Gene Name:||APAF1|