Figure 1. Induced phosphorylation of Aurora A in paclitaxel-treated Hela cells lysed in the presence of phosphatase inhibitors (phospho lysate) is detected by PathScan® Phospho-Aurora A (Thr288) Sandwich ELISA Kit #7114 (upper, right). In contrast, a low level of phospho-Aurora A protein is detected in paclitaxel-treated HeLa cells lysed without addition of phosphatase inhibitors to the lysis buffer (nonphospho lysate). Similar levels of Aurora A protein from either nonphospho or phospho lysates are detected by PathScan® Total Aurora A Sandwich ELISA Kit #7116 (upper, left). The absorbance readings at 450 nm are shown in the top figure, while the corresponding Western blots using Aurora A Antibody #3092 (left panel) or Phospho-Aurora A (Thr288) Rabbit mAb #3079 (right panel) are shown in the bottom figure.
Figure 2: The relationship between protein concentration of phospho or nonphospho Aurora A lysates and the absorbance at 450 nm is shown. HeLa cells (85% confluence) treated with paclitaxel (100 nM) for 20 hours were harvested and then lysed in the absence or presence of phosphatase inhibitor.
The PathScan® Total Aurora A Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of total Aurora A protein. An Aurora A Rabbit Antibody has been coated onto the microwells. After incubation with cell lysates, Aurora A (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, an Aurora A Mouse Detection Antibody is added to detect the captured phospho and nonphospho Aurora A protein. Anti-mouse IgG, HRP-linked Antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of total Aurora A protein.
Antibodies in kit are custom formulations specific to kit.
CST's PathScan® Total Aurora A Sandwich ELISA Kit #7116 detects endogenous levels of total Aurora A proteins. As shown in Figure 1, a significant induction of Phospho-Aurora A (Thr288) can be detected in HeLa cells treated with paclitaxel using the Phospho-Aurora A (Thr288) Sandwich ELISA Kit #7114. These high levels are abolished when paclitaxel-treated HeLa cells were lysed without addition of phosphatase inhibitors* to the lysis buffer. The levels of total Aurora A protein (either phospho or nonphospho) detected by PathScan® Total Aurora A Sandwich ELISA Kit #7116 remain unchanged during the G2/M phase of the cell cycle. Western analysis of protein captured in microwells coated with Aurora A antibody shows a single band corresponding to Aurora A protein (data not shown). This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
* Phosphatase inhibitors include sodium pyrophosphate, β-glycerophosphate and Na3VO4.
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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