Product Pathways - PathScan ELISA
PathScan® Phospho-Aurora A (Thr288) Sandwich ELISA Kit #7114
| Kit Includes | Volume | Solution Color |
|---|---|---|
| Aurora A Rabbit Antibody Coated Microwells | 96 tests | |
| Biotinylated Phospho-Aurora A (Thr288) Rabbit Detection Antibody | 11 milliliters | Green |
| HRP-linked Streptavidin | 11 milliliters | Red |
| TMB Substrate | 11 milliliters | Colorless |
| STOP Solution | 11 milliliters | Colorless |
| Sealing Tape | 2 sheets | |
| 20X Wash Buffer | 25 milliliters | Colorless |
| Sample Diluent | 25 milliliters | Blue |
| Cell Lysis Buffer (10X) # 9803 | 15 milliliters | Yellowish |
Note: 12 8-well modules –Each module is designed to break apart for 8 tests.
Note: Kit should be stored at 4°C with the exception of Cell Lysis Buffer (10X), which is stored at –20°C (packaged separately).
Species Cross-Reactivity
H
Reactivity Key: H=Human
Description
The PathScan® Phospho-Aurora A (Thr288) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Aurora A when Thr288 is phosphorylated. 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, a Biotinylated Phospho-Aurora A (Thr288) Rabbit Detection Antibody* is added to detect threonine phosphorylation of the captured Aurora A protein. HRP-linked Streptavidin* 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 Aurora A phosphorylated on Thr288.* Antibodies in kit are custom formulations specific to kit.
Specificity / Sensitivity
CST's PathScan® Phospho-Aurora A (Thr288) Sandwich ELISA Kit #7114 detects Aurora A when Thr288 is phosphorylated during G2/M phase of the cell cycle. 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.* Phosphatase inhibitors include sodium pyrophosphate, β-glycerophosphate and Na3VO4.
Sandwich ELISA
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.
Sandwich ELISA
Figure 2: The relationship between protein concentration of phospho or nonphospho Aurora A lysates and the absorbance at 450 nm is shown. Unstarved 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.
Background
Aurora kinases belong to a highly conserved family of mitotic serine/threonine kinases with three members identified among mammals: Aurora A, Aurora B and Aurora C (1,2). Studies on the temporal expression pattern and subcellular localization of Aurora kinases in mitotic cells suggest an association with mitotic structure. Their functional influences span from G2 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, while 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), while overexpression of both kinases is seen 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, overexpression of Aurora C is detected in various cancer cell lines (6).
- Warner, S.L. et al. (2003) Mol. Cancer Ther. 2, 589-595.
- Katayama , H. et al. (2003) Cancer Metastasis Rev. 22, 451-464.
- Andrews, P.D. et al. (2003) Curr. Opin. Cell Biol. 15, 672-683.
- Pascreau, G. et al. (2003) Prog. Cell Cycle Res. 5, 369-374.
- Crosio, C. et al. (2002) Mol. Cell. Biol. 22, 874-885.
- Kimura, M. et al. (1999) J. Biol. Chem. 274, 7334-7340.
Application References
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Companion Products
- 4718 Aurora A/AIK (1G4) Rabbit mAb
- 3092 Aurora A/AIK Antibody
- 2914 Phospho-Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (Thr198) (D13A11) Rabbit mAb
- 9803 Cell Lysis Buffer (10X)
- 9808 Phosphate Buffered Saline (PBS-20X)
- 9809 Phosphate Buffered Saline with Tween 20 (PBST-20X)
- 9998 BSA
- 7004 TMB Substrate
- 7002 STOP Solution
This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.
