Mouse Eukaryotic Translation Initiation Factor 2 Alpha Kinase 3 (EIF2aK3) ELISA Kit

Principle of the Assay

The microtiter plate provided in this kit has been pre-coated with an antibody specific to EIF2aK3. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific to EIF2aK3. 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 EIF2aK3, 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 EIF2aK3 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

Metabolic-stress sensing protein kinase that phosphorylates the alpha subunit of eukaryotic translation initiation factor 2 (EIF2S1/eIF-2-alpha) in response to various stress conditions (PubMed:9930704, PubMed:11106749, PubMed:23921556). Key activator of the integrated stress response (ISR) required for adaptation to various stress, such as unfolded protein response (UPR) and low amino acid availability (PubMed:9930704, PubMed:11106749, PubMed:23921556). EIF2S1/eIF-2-alpha phosphorylation in response to stress converts EIF2S1/eIF-2-alpha in a global protein synthesis inhibitor, leading to a global attenuation of cap-dependent translation, while concomitantly initiating the preferential translation of ISR-specific mRNAs, such as the transcriptional activators ATF4 and QRICH1, and hence allowing ATF4- and QRICH1-mediated reprogramming (PubMed:11106749, PubMed:23921556). Serves as a critical effector of unfolded protein response (UPR)-induced G1 growth arrest due to the loss of cyclin-D1 (CCND1) (PubMed:11035797). Involved in control of mitochondrial morphology and function (PubMed:23921556).

GENE ID	13666
SWISS PROT	Q9Z2B5
SYNONYMS	WRS; PEK; PERK; HsPEK; PRKR-like endoplasmic reticulum kinase; Pancreatic eIF2-alpha kinase

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 EIF2aK3 were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays): 3 samples with low, middle, and high-level EIF2aK3 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 0.108ng/mL.
ASSAY RANGE	0.312-20ng/mL
SPECIFICITY	This assay has high sensitivity and excellent specificity for the detection of EIF2aK3. No significant cross-reactivity or interference between EIF2aK3 and analogs was observed. Note: Limited by current skills and knowledge, it is impossible to perform all possible cross-reactivity detection tests between EIF2aK3 and all analogs, therefore, cross reactivity may still exist.