RAD17 antibody

Catalog No. ABIN492574

Product Details anti-RAD17 Antibody

Target: RAD17

Reactivity: Human, Mouse, Rat

Host: Mouse

Clonality: Monoclonal

Conjugate: This RAD17 antibody is un-conjugated

Application: Western Blotting (WB), Immunofluorescence (IF), Immunohistochemistry (Paraffin-embedded Sections) (IHC (p)), Immunoprecipitation (IP)

Specificity: This antibody reacts with HRad17 (80 kDa) on Western blotting, Immunoprecipitation and Immunohistochemistry.

Cross-Reactivity (Details): Species reactivity (tested):Human, Mouse and Rat.

Characteristics: Synonyms: R24L, Cell cycle checkpoint protein RAD17, RF-C/activator 1 homolog

Purification: Protein-A Sepharose Chromatography of hybridoma supernatant.

Immunogen: Recombinant full-length Human HRad17. Remarks: Hybridoma was established by fusion of mouse myeloma cell Sp2/0-Ag14 withBalb/c mouse splenocyte

Clone: 3-10E-010

Isotype: IgG1

Application Details

Application Notes: Western blotting: 1 μg/mL for chemiluminescence detection system. Immunoprecipitation: 2 μg/200 μL of cell extract from 5x10^6 cells. Immunohistochemistry on Paraffin Embedded Sections: 1-5 μg/mL (Heat treatment isnecessary). Microwave oven: 2 times for 10 minutes each in 10 mM citrate buffer ( pH 6.5). Detailed procedure is provided in Protocols.
Other applications not tested.
Optimal dilutions are dependent on conditions and should be determined by the user.

Protocol: SDS-PAGE & Western Blotting1) Wash the cells 3 times with PBS and suspend with 10 volume of cold Lysis buffer (50 mMTris-HCl, pH 7. 2, 250 mM NaCl, 0. 1% NP-40, 2 mM EDTA, 10%glycerol) containingappropriate protease inhibitors. Incubate it at 4°C with rotating for 30 minutes, thensonicate briefly (up to 10 seconds). 2) Centrifuge the tube at 12,000 x g for 10 minutes at 4°C and transfer the supernatant toanother tube. Measure the protein concentration of the supernatant and add the cold Lysisbuffer to make 8 mg/mL solution. 3) Mix the sample with equal volume of Laemmli's sample buffer. 4) Boil the samples for 3 minutes and centrifuge. Load 10 μL of the sample per lane in a 1mm thick SDS-polyacrylamide gel for electrophoresis. 5) Blot the protein to a polyvinylidene difluoride (PVDF) membrane at 1 mA/cm2 for 1 hourin a semi-dry transfer system (Transfer Buffer: 25 mM Tris, 190 mM glycine, 20% MeOH). See the manufacture's manual for precise transfer procedure. 6) To reduce nonspecific binding, soak the membrane in 10% skimmed milk (in PBS, pH7. 2) for 1 hour at room temperature, or overnight at 4°C. 7) Incubate the membrane with primary antibody diluted with PBS, pH 7. 2 containing 1%skimmed milk as suggest in the APPLICATIONS for 1 hour at room temperature. (Theconcentration of antibody will depend on condition. )8) Wash the membrane with PBS-T [0. 05% Tween-20 in PBS] (5 minutes x 3 times). 9) Incubate the membrane with the 1: 10,000 HRP-conjugated anti-mouse IgG diluted with1% skimmed milk (in PBS, pH 7. 2) for 1 hour at room temperature. 10) Wash the membrane with PBS-T (10 minutes x 3 times). 11) Wipe excess buffer on the membrane, then incubate it with appropriatechemiluminescence reagent for 1 minute. Remove extra reagent from the membrane by dabbing with paper towel, and seal it inplastic wrap. 12) Expose to an X-ray film in a dark room for 3 minutes. Develop the film as usual. The condition for exposure and development may vary. Positive Controls: HeLa, HEp-2, NIH/3T3, PC12Immunoprecipitation1) Wash the cells 3 times with PBS and suspend with 10 volume of cold Lysis buffer (50 mMTris-HCl pH 7. 2, 250 mM NaCl, 0. 1% NP-40, 2 mM EDTA, 10% glycerol) containingappropriate protease inhibitors. Incubate it at 4°C with rotating for 30 minutes, thensonicate briefly (up to 10 seconds). 2) Centrifuge the tube at 12,000 x g for 10 minutes at 4°C and transfer the supernatant toanother tube. 3) Add primary antibody as suggest in the APPLICATIONS into 200 μL of the supernatant. Mix well and incubate with gentle agitation for 30-120 minutes at 4°C. Add 20 μL of 50%protein A agarose resuspended in the cold Lysis buffer. Mix well and incubate with gentleagitation for 60 minutes at 4°C. 4) Wash the beads 3-5 times with the cold Lysis buffer (centrifuge the tube at 2,500 x g for

Restrictions: For Research Use only

Handling

Concentration: 1.0 mg/mL

Buffer: PBS, pH 7.2 containing 50 % Glycerol without preservatives.

Preservative: Without preservative

Storage: -20 °C

Storage Comment: Store the antibody (in aliquots) at -20 °C. Avoid repeated freezing and thawing.
Shelf life: one year from despatch.

Expiry Date: 12 months

Target Details for RAD17

Target: RAD17

Alternative Name: RAD17 (RAD17 Products)

Synonyms: zgc:91969 antibody, RAD17 antibody, K2A18.21 antibody, K2A18_21 antibody, RADIATION SENSITIVE 17 antibody, CCYC antibody, HRAD17 antibody, R24L antibody, RAD17SP antibody, RAD24 antibody, 9430035O09Rik antibody, MmRad24 antibody, RAD17 checkpoint clamp loader component antibody, Rad17p antibody, RADIATION SENSITIVE 17 antibody, RAD17 antibody, rad17 antibody, ATRAD17 antibody, Rad17 antibody

Background: HRad17 is the human homologue of the protein encoded by the yeast rad17 gene. The rad17 gene of Schizosaccharomyces pombe is one of several essential checkpoint components including rad1, rad3, rad9, rad17, hus1 and cut5/rad4, these are absolutely required for prevention of mitosis after DNA damage in fission yeast. HRad17 is localized to chromosome 5q12, 13.1, a region known to be deleted in a variety of human cancers which could lead to higher rate of mutation as well as increased sensitivity to radiation and chemotherapy.Synonyms: Cell cycle checkpoint protein RAD17, R24L, RF-C/activator 1 homolog

Gene ID: 5884

UniProt: O75943