Human Centromere Protein E (CENPE) ELISA Kit

Principle of the Assay

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

Microtubule plus-end-directed kinetochore motor which plays an important role in chromosome congression, microtubule-kinetochore conjugation and spindle assembly checkpoint activation. Drives chromosome congression (alignment of chromosomes at the spindle equator resulting in the formation of the metaphase plate) by mediating the lateral sliding of polar chromosomes along spindle microtubules towards the spindle equator and by aiding the establishment and maintenance of connections between kinetochores and spindle microtubules (PubMed:7889940, PubMed:23891108, PubMed:25395579). The transport of pole-proximal chromosomes towards the spindle equator is favored by microtubule tracks that are detyrosinated (PubMed:25908662). Acts as a processive bi-directional tracker of dynamic microtubule tips; after chromosomes have congressed, continues to play an active role at kinetochores, enhancing their links with dynamic microtubule ends (PubMed:23955301). Suppresses chromosome congression in NDC80-depleted cells and contributes positively to congression only when microtubules are stabilized (PubMed:25743205). Plays an important role in the formation of stable attachments between kinetochores and spindle microtubules (PubMed:17535814) The stabilization of kinetochore-microtubule attachment also requires CENPE-dependent localization of other proteins to the kinetochore including BUB1B, MAD1 and MAD2. Plays a role in spindle assembly checkpoint activation (SAC) via its interaction with BUB1B resulting in the activation of its kinase activity, which is important for activating SAC. Necessary for the mitotic checkpoint signal at individual kinetochores to prevent aneuploidy due to single chromosome loss (By similarity).

GENE ID	1062
SWISS PROT	Q02224
SYNONYMS	CENP-E; KIF10; Centromere-Associated Protein E

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 CENPE were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays): 3 samples with low, middle, and high-level CENPE 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 0.124ng/mL.
ASSAY RANGE	0.312-20ng/mL
SPECIFICITY	This assay has high sensitivity and excellent specificity for the detection of CENPE. No significant cross-reactivity or interference between CENPE and analogs was observed. Note: Limited by current skills and knowledge, it is impossible to perform all possible cross-reactivity detection tests between CENPE and all analogs, therefore, cross reactivity may still exist.