Figure 1. Treatment of HeLa cells with UV stimulates phosphorylation of Chk1 at Ser345, as detected by the PathScan® Phospho-Chk1 (Ser345) Sandwich ELISA Kit, but does not affect the levels of total Chk1 detected by the PathScan® Total Chk1 Sandwich ELISA Kit #7872. 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 (Ser345) Rabbit mAb #2348 (right panel) are shown in the bottom figure.
Figure 2. The relationship between the protein concentration of lysates from untreated and UV-treated HeLa cells and the absorbance at 450 nm using the PathScan® Phospho-Chk1 (Ser345) Sandwich ELISA Kit is shown. HeLa cells (80-90% confluent) were treated with 100 mJ/cm2 UV with 1 hour recovery at 37ºC and then lysed with the lysis buffer.
|Product Includes||Volume||Solution Color|
|Chk1 Mouse mAb Coated Microwells||96 tests|
|Phospho-Chk1 Ser345 Rabbit Detection mAb||1 ea|
|Anti-rabbit IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) 9803||15 ml|
The PathScan® Phospho-Chk1 (Ser345) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Chk1 when phosphorylated at Ser345. A 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 (Ser345) Rabbit Detection Antibody is added to detect phosphorylation of Ser345 on the captured Chk1 protein. Anti-rabbit 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 Chk1 phosphorylated at Ser345.
Antibodies in kit are custom formulations specific to the kit.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
Protocol Id: 204
PathScan® Phospho-Chk1 (Ser345) Sandwich ELISA Kit detects endogenous levels of Chk1 protein when phosphorylated at Ser345, as shown in Figure 1.
The kit sensitivity is shown in Figure 2. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
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 by ATM/ATR, followed by autophosphorylation of Ser296. Activation 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).
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