Product Pathways - PathScan ELISA
PathScan® Phospho-Chk1 (Ser317) Sandwich ELISA Kit #7870
|7870S||1 Kit (96 assays)||---||In Stock||---|
|7870||carrier free and custom formulation / quantity||email request|
When ordering five or more kits, please contact us for processing time and pricing at firstname.lastname@example.org.
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|Kit Includes||Volume||Solution Color|
|Chk1 Ab Coated Microwells||96 tests|
|P-Chk1 (S317) Detection Ab||11 ml||Green|
|Anti-rabbit IgG, HRP-linked Antibody||11 ml||Red|
|TMB Substrate #7004||11 ml||Colorless|
|STOP Solution #7002||11 ml||Colorless|
|Sealing Tape||2 sheets|
|ELISA Wash Buffer (20X)||25 ml||Colorless|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) #9803||15 ml||Yellowish|
Note: 12 8-well modules – Each module is designed to break apart for 8 tests.
Storage: Kit should be stored at 4°C with the exception of Cell Lysis Buffer, which is stored at –20°C (packaged separately).
The PathScan® Phospho-Chk1 (Ser317) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Chk1 when phosphorylated at Ser317. An Chk1 Mouse Antibody has been coated onto the microwells. After incubation with cell lysates, Chk1 (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, a Phospho-Chk1 (Ser317) Rabbit Detection Antibody is added to detect phosphorylation of Ser317 on the captured Chk1 protein. Anti-rabbit IgG, HRP-linked Antibody #7074 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 Chk1 phosphorylated at Ser317.
Antibodies in kit are custom formulations specific to kit.
Specificity / Sensitivity
CST's PathScan® Phospho-Chk1 (Ser317) Sandwich ELISA Kit #7870 detects endogenous levels of Chk1 protein when phosphorylated at Ser317. As shown in Figure 1, a significant induction of Chk1 phosphorylation at Ser317 can be detected in HeLa cells following treatment with UV using the Phospho-Chk1 (Ser317) Sandwich ELISA Kit #7870. The levels of total Chk1 (phospho and nonphospho) remain unchanged as shown by Western analysis and by PathScan® Total Chk1 Sandwich ELISA Kit #7872 (Figure 1). This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
ELISA - Western correlation
Figure 1. Treatment of HeLa cells with UV stimulates phosphorylation of Chk1 at Ser317, detected by the PathScan® Phospho-Chk1 (Ser317) Sandwich ELISA Kit #7870, but does not affect the levels of total Chk1 detected by PathScan® Total Chk1 Sandwich ELISA Kit #7872. HeLa cells (80-90% confluent) were treated with 100 mJ/cm2 UV with 1 hour recovery at 37ºC. The absorbance readings at 450 nm are shown in the top figure, while the corresponding Western blots using Chk1 Antibody #2345 (left panel) or Phospho-Chk1 (Ser317) Antibody #2344 (right panel) are shown in the bottom figure.
Chk1 kinase acts downstream of ATM/ATR kinase and plays an important role in DNA damage checkpoint control, embryonic development, and tumor suppression (1). Activation of Chk1 involves phosphorylation at Ser317 and Ser345 and occurs in response to blocked DNA replication and certain forms of genotoxic stress (2). While phosphorylation at Ser345 serves to localize Chk1 to the nucleus following checkpoint activation (3), phosphorylation at Ser317 along with site-specific phosphorylation of PTEN allows for re-entry into the cell cycle following stalled DNA replication (4). Chk1 exerts its checkpoint mechanism on the cell cycle, in part, by regulating the cdc25 family of phosphatases. Chk1 phosphorylation of cdc25A targets it for proteolysis and inhibits its activity through 14-3-3 binding (5). Activated Chk1 can inactivate cdc25C via phosphorylation at Ser216, blocking the activation of cdc2 and transition into mitosis (6). Centrosomal Chk1 has been shown to phosphorylate cdc25B and inhibit its activation of CDK1-cyclin B1, thereby abrogating mitotic spindle formation and chromatin condensation (7). Furthermore, Chk1 plays a role in spindle checkpoint function through regulation of aurora B and BubR1 (8). Research studies have implicated Chk1 as a drug target for cancer therapy as its inhibition leads to cell death in many cancer cell lines (9).
- Liu, Q. et al. (2000) Genes Dev 14, 1448-59.
- Zhao, H. and Piwnica-Worms, H. (2001) Mol Cell Biol 21, 4129-39.
- Jiang, K. et al. (2003) J Biol Chem 278, 25207-17.
- Martin, S.A. and Ouchi, T. (2008) Mol Cancer Ther 7, 2509-16.
- Chen, M.S. et al. (2003) Mol Cell Biol 23, 7488-97.
- Zeng, Y. et al. (1998) Nature 395, 507-10.
- Löffler, H. et al. (2006) Cell Cycle 5, 2543-7.
- Zachos, G. et al. (2007) Dev Cell 12, 247-60.
- Garber, K. (2005) J Natl Cancer Inst 97, 1026-8.
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- 7872 PathScan® Total Chk1 Sandwich ELISA Kit
- 12302 Phospho-Chk1 (Ser317) (D12H3) XP® Rabbit mAb
- 8191 Phospho-Chk1 (Ser317) (D7H2) Rabbit mAb
- 2344 Phospho-Chk1 (Ser317) Antibody
- 2345 Chk1 Antibody
- 2360 Chk1 (2G1D5) Mouse mAb
- 7074 Anti-rabbit IgG, HRP-linked Antibody
- 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
For Research Use Only. Not For Use In Diagnostic Procedures.
PathScan® is a trademark of Cell Signaling Technology, Inc.
Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.