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-Chk2 (Thr68) Sandwich ELISA Kit #7037 is shown.
Figure 1. Treatment of HeLa cells with UV stimulates phosphorylation of Chk2 at Thr68, detected by the PathScan® Phospho-Chk2 (Thr68) Sandwich ELISA Kit #7037, but does not affect the levels of total Chk2 detected by PathScan® Total Chk2 Sandwich ELISA Kit #7045. HeLa cells (80-90% confluent) were treated with 100 mJ/cm2 UV with 1 hour recovery at 37º C. The absorbance readings at 450 nm are shown in the top figure, while the corresponding western blots using Chk2 (1C12) Mouse mAb #3440 (left panel) or Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb #2197 (right panel) are shown in the bottom figure.
|7037C||1 Kit (96 assays)||$ 513|
|Product Includes||Volume||Solution Color|
|Phospho-Chk2 (Thr68) Rabbit mAb Coated Microwells||96 tests|
|Chk2 Mouse Detection mAb||1 ea||Green (Lyophilized)|
|Anti-mouse 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-Chk2 (Thr68) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Chk2 when phosphorylated at Thr68. A phospho-Chk2 (Thr68) rabbit antibody has been coated onto the microwells. After incubation with cell lysates, phospho-Chk2 protein is captured by the coated antibody. Following extensive washing, a Chk2 mouse detection antibody is added to detect the captured Chk2 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 the developed color is proportional to the quantity of Chk2 phosphorylated at Thr68.
Antibodies in kit are custom formulations specific to 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
CST's PathScan® Phospho-Chk2 (Thr68) Sandwich ELISA Kit #7037 detects endogenous levels of Chk2 protein when phosphorylated at Thr68. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
Chk2 is the mammalian orthologue of the budding yeast Rad53 and fission yeast Cds1 checkpoint kinases (1-3). The amino-terminal domain of Chk2 contains a series of seven serine or threonine residues (Ser19, Thr26, Ser28, Ser33, Ser35, Ser50, and Thr68) each followed by glutamine (SQ or TQ motif). These are known to be preferred sites for phosphorylation by ATM/ATR kinases (4,5). After DNA damage by ionizing radiation (IR), UV irradiation, or hydroxyurea treatment, Thr68 and other sites in this region become phosphorylated by ATM/ATR (5-7). The SQ/TQ cluster domain, therefore, seems to have a regulatory function. Phosphorylation at Thr68 is a prerequisite for the subsequent activation step, which is attributable to autophosphorylation of Chk2 at residues Thr383 and Thr387 in the activation loop of the kinase domain (8).
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