CDC27 antibody (C-Term)

Catalog No. ABIN492595

This Mouse Monoclonal antibody specifically detects CDC27 in WB, IP, IHC (p) and EIA. It exhibits reactivity toward Human, Rat, Mouse, Dog, Cow and Xenopus laevis.

Quick Overview for CDC27 antibody (C-Term) (ABIN492595)

Target: CDC27 (Cell Division Cycle 27 Homolog (S. Cerevisiae) (CDC27))

Reactivity: Human, Rat, Mouse, Dog, Cow, Xenopus laevis

Host: Mouse

Clonality: Monoclonal

Conjugate: This CDC27 antibody is un-conjugated

Application: Western Blotting (WB), Immunoprecipitation (IP), Immunohistochemistry (Paraffin-embedded Sections) (IHC (p)), Enzyme Immunoassay (EIA)

Clone: AF3-1

Product Details anti-CDC27 Antibody

Binding Specificity: AA 814-823, C-Term

Specificity: This antibody reacts with 97 kDa membrane protein CDC27 on Western blotting, Immunoprecipitation and Immunohistochemistry.

Cross-Reactivity (Details): Species reactivity (expected):Bovine, Canine and Xenopus leavis.
Species reactivity (tested):Human, Mouse, Rat.

Characteristics: Synonyms: CDC27Hs, H-NUC, D0S1430E, D17S978E, Cell division cycle protein 27 homolog

Purification: Protein-A Agarose Chromatography of hybridoma supernatant.

Immunogen: Peptide corresponding to C-terminal of Human CDC27 (aa 814-823).

Isotype: IgG2b

Application Details

Application Notes: Western blotting: 1 μg/mL for chemiluminescence detection system. Positive Controls: Jurkat, Raji, K562, HL-60. Immunoprecipitation: 2 μg/200 μL of cell extract. Positive Control: Raji. Immunohistochemistry on Paraffin Embedded Sections: 10 μg/mL. (Heat treatment is necessary)Autoclave: 10 minutes at 110 °C in 10 mM citrate buffer ( pH 6.5). Positive Control: Tonsil. 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 RT. 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. 12) Remove extra reagent from the membrane by dabbing with paper towel, and seal it inplastic wrap. 13) Expose to an X-ray film in a dark room for 3 minutes. 14) Develop the film as usual. The condition for exposure and development may vary. Positive Controls: Jurkat, Raji, K562, HL-60, MCF-7Immunoprecipitation1) Wash the cells 3 x 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 suggested in the APPLICATIONS into 200 µL of the supernatant.

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 CDC27

Target: CDC27 (Cell Division Cycle 27 Homolog (S. Cerevisiae) (CDC27))

Alternative Name: CDC27

Background: CDC16, CDC27 and CDC34 are components of the APC (anaphase-promoting complex) that also play a role as components of the ubiquitin- conjugating E3 enzyme. A 20S complex containing CDC27 and CDC16 catalyzes the mitosis-specific conjugation of ubiquitin to cyclin B, resulting in cyclin B/Cdk complex degradation. CDC27 is therefore required for progression of the cell cycle, where it executes essential mitotic functions near the metaphase/anaphase transition. CDC27 is ~97 kDa and is localized to the centrosome and mitotic spindle.Synonyms: CDC27Hs, Cell division cycle protein 27 homolog, D0S1430E, D17S978E, H-NUC

Gene ID: 996

UniProt: P30260