Rad9 [p Ser1260] Antibody - BSA Free

Western Blot: Rad9 [p Ser1260] Antibody [NBP1-77981] - Affinity purified Rad9 [p Ser1260] antibody used at a 1:200 dilution incubated overnight at 4C to detect Rad9 by Western blot. Lanes loaded with 50 ng each of recombinant GST fusion protein containing S. cerevisiae Rad9 (aa 991-1309 ~60 kDa) on a 4-20% Criterion gel for SDS-PAGE as follows: Lane 1 - non-phosphorylated wild type yeast Rad9, Lane 2 - in vitro phosphorylated wild type yeast Rad9, Lane 3 - non-phosphorylated S1129A/S1260A double mutant Rad9, Lane 4 - in vitro phosphorylated S1129A/S1260A double mutant. Phosphorylation of Rad9 by treatment with ATP & Rad53 kinase. Rad53 kinase autophosphorylates & appears cross reactive as it is detected on the blot as 90 & 110 kDa bands (asterisk). Detection occurred using a 1:5,000 IRDye(TM)800 conjugated Donkey anti-Rabbit IgG for 1h at room temperature. LICOR's Odyssey(R) Infrared Imaging System was used to scan & process the image.

N/A: Rad9 [p Ser1260] Antibody [NBP1-77981]

• Reactivity: Ye
• Applications: WB, ELISA
• Clonality: Polyclonal
• Host: Rabbit
• Conjugate: Unconjugated
• Format: BSA Free

Rad9 [p Ser1260] Antibody - BSA Free Summary

• Description: 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 is phospho specific to yeast pS1260.
  
  Store vial at -20C prior to opening. Aliquot contents and freeze at -20C or below for extended storage. Avoid cycles of freezing and thawing. Centrifuge product if not completely clear after standing at room temperature. This product is stable for several weeks at 4C as an undiluted liquid. Dilute only prior to immediate use.
• Immunogen: This affinity purified Rad9 [p Ser1260] Antibody was prepared from whole rabbit serum produced by repeated immunizations with a synthetic peptide corresponding to phosphorylated form of an internal region near aa 1225-1275 from the aa1309 yeast Rad9 protein conjugated to KLH. (Uniprot: P14737)
• Modification: p Ser1260
• Specificity: This affinity purified antibody is directed against the phosphorylated form of yeast Rad9 at the pS1260 residue. The resultant affinity purified antibody is phospho specific to yeast pS1260.
• Isotype: IgG
• Clonality: Polyclonal
• Host: Rabbit
• Gene: RAD9A
• Purity: Immunogen affinity purified

Applications/Dilutions

• Dilutions:
  • ELISA 1:5000
  • Western Blot 1:500-1:2000
• Application Notes: 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. No reactivity is expected against the human or mouse analogs of RAD9. Reactivity against RAD9 from other sources is unknown. Cross reactivity may occur with auto-phosphorylated Rad53 kinase. Although not tested, this antibody is likely functional by IHC and IP. 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) 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.

Packaging, Storage & Formulations

• Storage: Store at -20C short term. Aliquot and store at -80C long term. Avoid freeze-thaw cycles.
• Buffer: 0.02 M Potassium Phosphate, 0.15 M Sodium Chloride, pH 7.2
• Preservative: 0.01% Sodium Azide
• Purity: Immunogen affinity purified

Alternate Names for Rad9 [p Ser1260] Antibody - BSA Free

• cell cycle checkpoint control protein RAD9A
• DNA repair exonuclease rad9 homolog A
• EC 3.1.11.2
• hRAD9
• RAD9 (S. pombe) homolog
• RAD9 homolog A (S. pombe)
• RAD9

Background

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 G2/M arrest after irradiation with X-rays. Subsequently, it has become clear that Rad9 also functions at the G1/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. Rad9 conveys the checkpoint signal by activating Rad53p and Chk1p; is hyperphos-phorylated by Mec1p and Tel1p; and is a potential Cdc28p substrate. Mature yeast Rad9 is reported to have an apparent molecular weight of ~148kDa. The human homolog is reported at 48.5 kDa.

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Bioinformatics

• Gene Symbol: RAD9A
• Uniprot:
  • NP_004575(Human)
  • P14737(Yeast)