Technical Note: R&D Systems Antibodies & ELISA Kits for Cell Cycle Checkpoint Research
Cell cycle progression is regulated by a series of checkpoints that monitor the integrity of the DNA. When DNA damage is present, checkpoints are activated to slow or arrest the cell cycle and promote DNA repair or apoptosis. Cell cycle checkpoints rely on sensor proteins that bind to damaged DNA and initiate the recruitment of several other proteins to the damage site. Some of these proteins are responsible for maintaining checkpoint activation as long as DNA damage persists, while others transduce the damage signal by phosphorylating multiple downstream effector proteins. These phosphorylation events directly inhibit the transition from one phase of the cell cycle to the next. Mutations in cell cycle checkpoint proteins are associated with genomic instability syndromes and a predisposition to multiple types of cancer. Research is currently being conducted to reveal the functions of checkpoint proteins and the effects of critical phosphorylation events. This will provide insight into the mechanisms by which checkpoints are regulated and maintained. R&D Systems offers a wide selection of phospho-specific and total protein antibodies and ELISAs for the characterization of cell cycle checkpoint pathways (Table 1). For more information on products for cell cycle- and checkpoint-related research, please visit our website at www.RnDSystems.com/go/Genotoxic.
| Table 1. | |||
|---|---|---|---|
| Molecule | Antibodies | Antibody Applications | ELISAs/Kinase Activity Assays |
| Phospho-53BP1 (S25) | H | WB | |
| Total 53BP1 | H | WB | |
| Phospho-ATM (S1981) | H M R | WB | H |
| Total ATR | H | WB | |
| Total ATRIP | H | WB | |
| Phospho-BRCA1 (S1423) | H | IHC, WB | |
| Total BRCA1 | H M R | IHC, IP, WB | |
| Phospho-Chk1 (S317) | H M R | WB | |
| Phospho-Chk1 (S345) | H | WB | |
| Total Chk1 | H | IP, WB | H/M/R |
| Phospho-Chk2 (T68) | H | IHC, WB | H |
| Total Chk2 | H M R | IHC, WB | H/M/R |
| Total Claspin | H | IHC, WB | |
| Phospho-H2AX (S139) | H | IHC, WB | H/M/R |
| Total H2AX | H M R | WB | |
| Total Mre11 | H | WB | |
| Phospho-Nbs1 (S343) | H | WB | |
| Total Nbs1 | H M R | IP, WB | |
| Phospho-p53 (S15) | H | FC, IHC, IP, WB | H |
| Phospho-p53 (S18) | M | WB | |
| Phospho-p53 (S20) | H | WB | |
| Phospho-p53 (S37) | H | WB | |
| Phospho-p53 (S46) | H | IHC, WB | H |
| Phospho-p53 (S392) | H | WB | H |
| Total p53 | H M R | FC, IP, WB | H M |
| Phospho-Rad17 (S635) | H | WB | H |
| Total Rad17 | H M R | IP, WB | H |
| Total Rad50 | H | WB | |
| Phospho-SMC1 (S966) | H | WB | |
| Total SMC1 | H M R | WB | |
| Species Key: H Human, M Mouse, R Rat Antibody Application Key: FC Flow Cytometry, IP Immunoprecipitation, IHC Immunohistochemistry, WB Western Blot | |||
Detection of Phosphorylated Nbs1 on S343 by Western Blot. Exponentially growing HeLa human cervical epithelial carcinoma cells were mock-treated or treated with UV light. Nbs1 was immunoprecipitated from whole cell extracts prepared 3 hours after irradiation using anti-human Nbs1 polyclonal antibody (Catalog # AF1573) and Protein G agarose beads. Following elution, immunoprecipitates (IP) were immunoblotted alongside whole cell extracts (WCE) using anti-human Phospho-Nbs1 (S343) polyclonal antibody (Catalog # AF4944; top panel). The same membrane was stripped and reprobed with anti-human/mouse/rat Nbs1 monoclonal antibody (Catalog # MAB1573; bottom panel)
Detection of Phosphorylated p53 on S15 by Flow Cytometry. MCF-7 human breast cancer cells, untreated (light burgundy histogram) or treated with camptothecin (burgundy histogram), were incubated with anti-human Phospho-p53 (S15) monoclonal antibody (Catalog # MAB18391) or isotope control antibody (Catalog # MAB002; open histogram) followed by staining with PE-conjugated anti-mouse IgG (Catalog # F0102B).
Detection of Phosphorylated p53 on S37 by Western Blot. MCF-7 human breast cancer cells were mock-treated or treated with camptothecin (CPT) for 5 hours. Whole cell extracts were prepared and the indicated sample was treated with lambda phosphatase. p53 was immunoprecipitated from the extracts using anti-human/mouse/rat p53 agarose (Catalog # GAF1355). Following elution, immunoprecipitated proteins were immunoblotted using anti-human Phospho-p53 (S37) polyclonal antibody (Catalog # AF3306; top panel). The same membrane was stripped and reprobed with HRP-conjugated anti-human/mouse/rat p53 polyclonal antibody (Catalog # HAF1355; bottom panel).