Separase antibody (AA 1866-1966)

Catalog No. ABIN5540331

The Mouse Monoclonal anti-Separase antibody has been validated for WB. It is suitable to detect Separase in samples from Human.

Quick Overview for Separase antibody (AA 1866-1966) (ABIN5540331)

Target: Separase (ESPL1) (Extra Spindle Poles Like 1 (ESPL1))

Reactivity: Human

Host: Mouse

Clonality: Monoclonal

Conjugate: This Separase antibody is un-conjugated

Application: Western Blotting (WB)

Clone: XJ11-1B12

Product Details anti-Separase Antibody

Binding Specificity: AA 1866-1966

Specificity: This antibody reacts with human Separase (190 kDa).

Cross-Reactivity (Details): Does not work with Mouse (WR19L, U937, P19, NIH/3T3) and Hamster (CHO).

Purification: Protein A agarose

Immunogen: Separase (1866-1966 aa)

Isotype: IgG1

Application Details

Application Notes: Western blot: 1 μg/mL for chemiluminescence detection system. Not reconmmended for Immunoprecipitation, Immunohistochemistry, and Immunocytochemistry.

Protocol: SDS-PAGE & Western Blotting 1) Wash the cells 3 times with PBS and suspend with 10 volume of cold Lysis buffer (50 mM Tris-HCl, pH 7.2, 250 mM NaCl, 0.1 % NP-40, 2 mM EDTA, 10 % glycerol) containing appropriate protease inhibitors. Incubate it at 4 o C with rotating for 30 minutes, then sonicate briefly (up to 10 seconds). 2) Centrifuge the tube at 12,000 x g for 10 minutes at 4 o C and transfer the supernatant to another tube. Measure the protein concentration of the supernatant and add the Lysis buffer to make 8 mg/mL solution. 3) Mix the sample with equal volume of Laemmli's sample buffer. 4) Boil the samples for 2 minutes and centrifuge. Load 10 μ L of the sample per lane in a 1 mm thick SDS-polyacrylamide gel for electrophoresis. 5) Blot the protein to a polyvinylidene difluoride (PVDF) membrane at 1 mA/cm 2 for 1 hour in 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, pH 7.2) for 1 hour at room temperature, or overnight at 4 o 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 ro om temperature. (The concentration 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 with 1 % skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature. 10) Wash the membrane with PBS-T (5 minutes x 6 times). 11) Wipe excess buffer on the membrane, then incubate it with appropriate chemilumin escence reagent for 1 minute. Remove extra reagent from the membrane by dabbing with paper towel, and seal it in plastic wrap. 12) Expose to an X-ray film in a dark room for 10 minutes. Develop the film as usual. The condition for exposure and development may vary. (Positive controls for Western blotting HeLa, Raji, ZR-75-1, MCF-7, HL-60, Jurkat)

Restrictions: For Research Use only

Handling

Format: Liquid

Buffer: PBS containing 50 % glycerol, pH 7.2. No preservative is contained.

Preservative: Azide free

Storage: -20 °C

Storage Comment: Upon receipt, store undiluted (in aliquots) at -20°C. Avoid repeated freezing and thawing. Shelf life: One year from despatch.

Target Details for Separase

Target: Separase (ESPL1) (Extra Spindle Poles Like 1 (ESPL1))

Alternative Name: separin,espl1

Background: Separase (190 kDa) is a cysteine protease that is essential for chromosome segregation. During cell division the sister chromatids are held together by the cohesin complex. At the onset of anaphase, separase cleaves the SCC1/RAD21 subunit of cohesin, allowing the chromatids to separate and divide with the cell. During most of the cell cycle, separase is inactivated by the securin/PDS-1 protein, which probably covers its active site. Separase is also down re gulated by self-cleavage to a ~65 kDa form. Separase can be used as a marker for centrosomes in mitotic cells.

UniProt: Q14674