Rat Apoptosis Inducing Factor (AIF) ELISA Kit

Principle of the Assay

The microtiter plate provided in this kit has been pre-coated with an antibody specific to AIF. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific to AIF. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After the TMB substrate solution is added, only those wells that contain AIF, biotin-conjugated antibody, and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution, and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of AIF in the samples is then determined by comparing the O.D. of the samples to the standard curve.

For Use with serum, plasma, and cell culture supernatants. For Research Use Only. Not for use in diagnostic procedures.

Target Information

Functions both as NADH oxidoreductase and as regulator of apoptosis (By similarity). In response to apoptotic stimuli, it is released from the mitochondrion intermembrane space into the cytosol and to the nucleus, where it functions as a proapoptotic factor in a caspase-independent pathway. The soluble form (AIFsol) found in the nucleus induces 'parthanatos' i.e. caspase-independent fragmentation of chromosomal DNA (PubMed:9989411). Binds to DNA in a sequence-independent manner. Interacts with EIF3G, and thereby inhibits the EIF3 machinery and protein synthesis, and activates caspase-7 to amplify apoptosis. Plays a critical role in caspase-independent, pyknotic cell death in hydrogen peroxide-exposed cells (By similarity). In contrast, participates in normal mitochondrial metabolism. Plays an important role in the regulation of respiratory chain biogenesis by interacting with CHCHD4 and controlling CHCHD4 mitochondrial import (PubMed:19447115).

GENE ID	26926
SWISS PROT	Q9Z0X1
SYNONYMS	PDCD8; Programmed Cell Death protein 8; Apoptosis-inducing factor 1, mitochondrial

Materials Supplied

Kit Components	96 Wells Quantity/Size
Pre-coated, ready-to-use 96-well strip plate	1 plate
Plate sealer for 96 wells	2
Standard	2 tubes
Diluent buffer	1 bottle
Detection Reagent A	1 bottle
Detection Reagent B	1 bottle
TMB Substrate	1 tube
Stop Solution	1 tube
Wash Buffer (30 ℅ concentrate)	1 tube
Product data sheet	1 copy

Storage

The TMB Substrate, Wash Buffer (30X concentrate), and the Stop Solution should be stored at 4°C upon receipt, while the other items should be stored at -20°C.

Performance Characteristics

REPEATABILITY	Intra-assay Precision (Precision within an assay): 3 samples with low, middle, and high-level AIF were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays): 3 samples with low, middle, and high-level AIF were tested on 3 different plates, with 8 replicates in each plate. CV(%) = SD/meanX100 Intra-Assay: CV<10% Inter-Assay: CV<12%
SENSITIVITY	The minimum detectable dose was 0.061ng/mL.
ASSAY RANGE	0.156-10ng/mL
SPECIFICITY	This assay has high sensitivity and excellent specificity for the detection of AIF. No significant cross-reactivity or interference between AIF and analogs was observed. Note: Limited by current skills and knowledge, it is impossible to perform all possible cross-reactivity detection tests between AIF and all analogs, therefore, cross reactivity may still exist.