Western blot analysis of CHK2 was performed by loading 25ug of the indicated cell lines and 10ul of PageRuler Prestained Protein Standard (Product # 26616) per well onto a Novex® 4-20% Tris-Glycine polyacrylamide gel (Product # WT4202). Proteins were transferred to a Nitrocellulose Membrane (Product # 88014) using the G2 Fast Blotter (Product # 62288), and blocked with 5% milk in TBST for at least 1 hour at room temperature. CHK2 was detected between 60-65kD using a CHK2 monoclonal antibody (Product # MA5-15416) at a concentration of 1ug/ml in blocking buffer overnight at 4C on a rocking platform, followed by an HRP-conjugated goat anti-mouse IgG secondary antibody (Product # 31430) at a dilution of 1:40,000 for at least 30 minutes at room temperature. Chemiluminescent detection was performed using SuperSignal West Dura (Product # 34076).
|Tested species reactivity||Human, Non-human primate|
|Host / Isotype||Mouse / IgG2b|
|Immunogen||Purified recombinant fragment of human CHK2 (aa481-531) expressed in E. Coli.|
|Storage buffer||PBS with 1mg/ml BSA, 30% glycerol|
|Contains||0.05% sodium azide|
|Tested Applications||Dilution *|
|Immunohistochemistry (IHC)||Assay dependent|
|Western Blot (WB)||0.5-2 µg/ml|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
CHK2 has a predicted MW of 61kD, and by Western blot MA5-15416 detects CHK2 between 60-65kD.
CHK2: CHK2 checkpoint homolog (S. pombe). In response to DNA damage and replication blocks, cell cycle progression is halted through the control of critical cell cycle regulators. The protein encoded by this gene is a cell cycle checkpoint regulator and putative tumor suppressor. It contains a forkhead-associated protein interaction domain essential for activation in response to DNA damage and is rapidly phosphorylated in response to replication blocks and DNA damage. When activated, the encoded protein is known to inhibit CDC25C phosphatase, preventing entry into mitosis, and has been shown to stabilize the tumor suppressor protein p53, leading to cell cycle arrest in G1. In addition, this protein interacts with and phosphorylates BRCA1, allowing BRCA1 to restore survival after DNA damage. Mutations in this gene have been linked with Li-Fraumeni syndrome, a highly penetrant familial cancer phenotype usually associated with inherited mutations in TP53. Also, mutations in this gene are thought to confer a predisposition to sarcomas, breast cancer, and brain tumors. This nuclear protein is a member of the CDS1 subfamily of serine/threonine protein kinases. Three transcript variants encoding different isoforms have been found for this gene.
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