|41333C||1 Kit (96 assays)||
|Product Includes||Volume||Solution Color|
|Phospho-Aurora A (Thr288) Rabbit mAb Coated Microwells||96 tests|
|Aurora A Rabbit Detection mAb||1 ea||Red (Lyophilized)|
|HRP Diluent||5.5 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X) 9801||25 ml|
|Cell Lysis Buffer (10X) 9803||15 ml|
The rapid protocol (RP) PathScan® RP Phospho-Aurora A (Thr288) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Aurora A protein phosphorylated at Thr288 in a reduced assay time of 1.5 hours. Incubation of cell lysates and detection antibody on the coated microwell plate forms a sandwich with phospho-Aurora A (Thr288) in a single step. The plate is then extensively washed and TMB reagent is added for signal development. The magnitude of absorbance for the developed color is proportional to the quantity of phospho-Aurora A (Thr288). Learn more about your ELISA kit options here.
*Antibodies in this kit are custom formulations specific to kit.
NOTE: This protocol is for PathScan® kits that use an HRP directly conjugated to the detection antibody (Rapid Protocol), rather than a 2-step method where the detection antibody and a secondary-HRP are added sequentially.
NOTE: Prepare solutions with deionized/purified water or equivalent.
For adherent cells
For suspension cells
NOTE: Equilibrate all materials and prepared reagents to room temperature prior to running the assay.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
created July 2020
Protocol Id: 2144
Aurora kinases belong to a highly conserved family of mitotic serine/threonine kinases with three members identified among mammals: Aurora A, B, and C (1,2). Studies on the temporal expression pattern and subcellular localization of Aurora kinases in mitotic cells suggest an association with mitotic structure. Aurora kinase functional influences span from G2 phase to cytokinesis and may be involved in key cell cycle events such as centrosome duplication, chromosome bi-orientation and segregation, cleavage furrow positioning, and ingression (3). Aurora A is detected at the centrosomes, along mitotic spindle microtubules, and in the cytoplasm of mitotically proliferating cells. Aurora A protein levels are low during G1 and S phases and peak during the G2/M phase of the cell cycle. Phosphorylation of Aurora A at Thr288 in its catalytic domain increases kinase activity. Aurora A is involved in centrosome separation, maturation, and spindle assembly and stability. Expression of Aurora B protein also peaks during the G2/M phase of the cell cycle; Aurora B kinase activity peaks at the transition from metaphase to the end of mitosis. Aurora B associates with chromosomes during prophase prior to relocalizing to the spindle at anaphase. Aurora B regulates chromosome segregation through the control of microtubule-kinetochore attachment and cytokinesis. Expression of both Aurora A and Aurora B during the G2/M phase transition is tightly coordinated with histone H3 phosphorylation (4,5); research investigators have observed overexpression of these kinases in a variety of human cancers (2,4). Aurora C localizes to the centrosome from anaphase to cytokinesis and both mRNA and protein levels peak during G2/M phase. Although typical Aurora C expression is limited to the testis, research studies report overexpression of Aurora C is detected in various cancer cell lines (6).
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