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Primary Antibodies  >  Phospho Specific Antibodies
 

Rad9 phospho S1260 Antibody

Rabbit Polyclonal
Code

600-401-468
Size

100 µg
Price

$309.00
Availability

Ships next business day.
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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 “Sensors”, which detect different sorts of damage, “Signal transducers” which are signal-integrating kinases, and “Targets” which carry out the essential functions of suppressing progress through the cell cycle (i.e. inducing repair genes and preventing late origin firing or sister chromatid segregation.  Contributed by C. Frei and K. Shimada, laboratory of S. Gasser, U. Geneva.
Affinity purified phospho-specific antibody to yeast Rad9 at pS1260 was used at a 1:200 dilution incubated overnight at 4° C to detect Rad9 by Western blot.  Lanes were 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 was by treatment with ATP and Rad53 kinase.  Rad53 kinase autophosphorylates and appears cross reactive as it is detected on the blot as 90 and 110 kDa bands (asterisk). Detection occurred using a 1:5,000 dilution of IRDye™800 conjugated Donkey anti-Rabbit IgG (code # 611-732-127) for 1h at room temperature. LICOR's Odyssey®  Infrared Imaging System was used to scan and process the image.  Other detection systems will yield similar results.
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Synonyms: Cell cycle checkpoint control protein antibody, Cell cycle checkpoint control protein RAD9A antibody, DNA repair exonuclease rad9 homolog A antibody

Rad9 phospho S1260 Antibody Properties

Description
Anti-Yeast Rad9 pS1260 (RABBIT) Antibody - 600-401-468
Host
Rabbit
Target Species
Yeast
Known Cross Reactivity
Saccharomyces cerevisiae
Clonality
Polyclonal
Application
ELISA : 1:5,000
Western Blot : 1:500- 1:2,000
Other Dilution: User Optimized
Physical State
Liquid (sterile filtered)
Shipping condition
Dry Ice
Format
IgG
Label
Concentration
0.41 mg/mL by UV absorbance at 280 nm
Buffer
0.02 M Potassium Phosphate, 0.15 M Sodium Chloride, pH 7.2
Preservative
0.01% (w/v) Sodium Azide
Stabilizer
None
GeneName
RAD9A
Download CofA
Lot#  

Rad9 phospho S1260 Antibody Description

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.
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.
Immunogen Type
Peptide
Post Translational Modification
Phosphorylation
Storage Condition
Store vial at -20° C prior to opening. Aliquot contents and freeze at -20° C 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 4° C as an undiluted liquid. Dilute only prior to immediate use.
Application 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. 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 µg 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.
Purity/Specifity
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.
Disclaimer Note-General
This product is for research use only and is not intended for therapeutic or diagnostic applications. Please contact a technical service representative for more information. All products of animal origin manufactured by Rockland Immunochemicals are derived from starting materials of North American origin. Collection was performed in United States Department of Agriculture (USDA) inspected facilities and all materials have been inspected and certified to be free of disease and suitable for exportation. All properties listed are typical characteristics and are not specifications. All suggestions and data are offered in good faith but without guarantee as conditions and methods of use of our products are beyond our control. All claims must be made within 30 days following the date of delivery. The prospective user must determine the suitability of our materials before adopting them on a commercial scale. Suggested uses of our products are not recommendations to use our products in violation of any patent or as a license under any patent of Rockland Immunochemicals, Inc. If you require a commercial license to use this material and do not have one, then return this material, unopened to: Rockland Inc., P.O. BOX 326, Gilbertsville, Pennsylvania, USA.
General Reference
de la Torre-Ruiz MA, et al. (1998) RAD9 and RAD24 define two additive, interacting branches of the DNA damage checkpoint pathway in budding yeast normally required for Rad53 modification and activation. EMBO J 17(9):2687-98. Fasullo, M., et al. (1998) The Saccharomyces cerevisiae RAD9 Checkpoint Reduces the DNA Damage-Associated Stimulation of Directed Translocations. Mol Cell Biol.;18 (3): 1190–1200. Sun, Z. et al. (1998) Rad53 FHA domain associated with phosphorylated Rad9 in the DNA damage checkpoint. Science 281: 272-274. Komatsu K., et al. (2000) Schizosaccharomyces pombe Rad9 contains a BH3-like region and interacts with the anti-apoptotic protein Bcl-2. FEBS Lett. 481(2):122-6. Schwartz M.F., et al. (2002) Rad9 Phosphorylation Sites Couple Rad53 to the Saccharomyces cerevisiae DNA Damage Checkpoint. Mol Cell 9(5):1055-65.
Specific Reference
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Product Type
Primary Antibodies;
Applications
ELISA, WB, IHC, IP, ChIP
Reacts With
human
Catalog Number
200-301-174

Product Type
Primary Antibodies;
Applications
ELISA, WB, IHC
Reacts With
human, mouse, rat
Catalog Number
200-306-400

Product Type
Primary Antibodies;
Applications
ELISA, WB
Reacts With
Saccharomyces cerevisiae
Catalog Number
600-401-345

Product Type
Primary Antibodies;
Applications
ELISA, WB
Reacts With
Saccharomyces cerevisiae
Catalog Number
600-401-467

Product Label

 

Description
Unconjugated
Color
Conjugation Reference
Molecular Weight
Excitation Wavelength
Conjugation Chemistry