RAD9 Homolog A (S. Pombe) (RAD9A) (pS1260) antibody

Antigen: RAD9 Homolog A (S. Pombe) (RAD9A)

Synonyms: RAD9, SMC1, SMCB, CDLS2, SB1.8, SMC1L1, DXS423E, KIAA0178, MGC138332, SMC1alpha, DKFZp686L19178, RAD9A, Rad9a, rad9a, MGC89062

Epitope: pS1260

Clonality: Polyclonal

Host: Rabbit

Reactivity: Yeast (Saccharomyces Cerevisiae)

Application: ELISA, Western Blotting (WB), Immunoprecipitation (IP)

Catalog no.: ABIN106405

Additional Information

Alternative name: Rad9 pS1260

Immunogen: This affinity purified antibody was prepared from whole rabbit serum produced by repeated immunizations with a synthetic peptide corresponding to phosphorylated form of aa 1249-1263 of 1309 of yeast Rad9 protein conjugated to KLH.

Format: Liquid (sterile filtered)

Description: Rad9 is required for the MEC1/TEL1-dependent activation of Saccharomyces cerevisiae DNA damage checkpoint pathways mediated by Rad53 and Chk1. DNA damage induces Rad9 phosphorylation, and Rad53 specifically associates with phosphorylated Rad9. Cells have evolved multiple strategies for tolerating genomic damage. The most important of these are numerous repair systems that remove or bypass potentially mutagenic DNA lesions. Another cellular strategy is to delay cell-cycle transitions at multiple points. The genetic control of these delays, termed `checkpoints', was first established in budding yeast where it was shown that the RAD9 gene functions in G 2 /M arrest after irradiation with X-rays. Subsequently, it has become clear that Rad9 also functions at the G 1 /S, intra-S and mid-anaphase checkpoints. Defects in checkpoint regulation can lead to genome instability and, in higher eukaryotes, neoplastic transformation. Rad9 also controls the transcriptional induction of a DNA damage regulon (DDR). Rad9 may also have a pro-apoptotic function. This is suggested in that Rad9 from Schizosaccharomyces pombe (SpRad9) contains a group of amino acids with similarity to the Bcl-2 homology 3 death domain, which is required for SpRad9 interaction with human Bcl-2 and apoptosis induction in human cells. Overexpression of Bcl-2 in S. pombe inhibits cell growth independently of rad9, but enhances resistance of rad9-null cells to methyl methanesulfonate, ultraviolet and ionizing radiation. Mature yeast Rad9 is reported to have an apparent molecular weight of ~148kDa. The human homolog is reported at 48.5 kDa.

Specificity: This affinity purified antibody is directed against the phosphorylated form of yeast Rad9 at the pS1260 residue. The product was affinity purified from monospecific antiserum by immunoaffinity purification. Antiserum was first purified against the phosphorylated form of the immunizing peptide. The resultant affinity purified antibody was then cross-adsorbed against the non-phosphorylated form of the immunizing peptide. This phospho specific polyclonal antibody reacts with phosphorylated pS1260 of yeast Rad9. Reactivity with non-phosphorylated yeast Rad9 is minimal by ELISA and immunoblotting. Cross reactivity with Rad9 homologs from other sources has not been determined.

Application Details

Application Notes: Recommended Dilutions: This product has been assayed against 0.1 ug of phosphorylated peptide (pS1260) in a standard capture ELISA using TMB (3,3',5,5'-Tetramethylbenizidine) code TMBE-100 as a substrate for 30 minutes at room temperature. A working dilution of 1:5,000 is suggested for this product. Minimal reactivity was detected against the non-phosphorylated form (S1260) of the immunizing peptide. This antibody appears to be specific for the active form (phosphorylated) of the protein. Dilute the antibody 1:100 to 1:500 for immunoblotting. Researchers should determine optimal titers for other applications. Note: This phospho specific polyclonal antibody was tested by immunoblotting and ELISA. Data from both immunoblotting and ELISA indicate the antibody is reactive with the phosphorylated form of the immunizing peptide and minimally reactive with the non-phosphorylated form of the immunizing peptide. Immunoblotting detects yeast Rad9 protein with unknown reactivity to human and mouse homologs. Cross reactivity may occur with auto-phosphorylated Rad53 kinase. Although not tested, this antibody is likely functional by IHC and IP.

Concentration: 0.41 mg/ml (by UV absorbance at 280 nm)

Buffer: Stabilizer: None. Preservative: 0.01% (w/v) Sodium Azide. Buffer: 0.02 M Potassium Phosphate, 0.15 M Sodium Chloride, pH 7.2.

Storage: Store vial at -20° C prior to opening. Centrifuge product if not completely clear after standing at room temperature. Dilute only prior to immediate use. For extended storage aliquot contents and freeze at -20° C or below. Avoid cycles of freezing and thawing. Expiration date is one (1) year from date of opening.

Restrictions: For Research Use only

Images

Immunoblotting. Affinity purified phosphospecific antibody to yeast Rad9 at pS1260 was used at a 1:200 dilution incubated overnight at 4

Summary Table. Checkpoints are mechanisms that impose delays in the cell cycle in response to DNA damage or defects in DNA replication, to ensure that mitotic transmission is error-free. Failure to delay the cell cycle in the presence of damage converts an easily reparable DNA lesion into one far more deleterious, provoking genomic instability or cell death. This figure shows a summary of our current knowledge about the DNA damage checkpoints in yeast. Genetic analysis of the pathway has allowed classification of its components into