Centromere Protein E, 312kDa (CENPE) antibody

Antigen: Centromere Protein E, 312kDa (CENPE)

Synonyms: KIF10, CENPE, kif10, cenp-E, XCENP-E

Clonality: Monoclonal (1H12)

Host: Mouse

Reactivity: Human

Application: Immunocytochemistry (ICC), Immunofluorescence (IF), Immunoprecipitation (IP), Western Blotting (WB)

Catalog no.: ABIN112051

Additional Information

Alternative name: CENPE

UniProt: Q02224

Immunogen: Recombinant human CENP-E.

Cross-Reactivity: Human

Isotype: IgG1

Clone: 1H12

Description: CENPE is a 250-300 kDa human centromere-associated kinesin-like motor protein thataccumulates in G2 phase. In contrast to other centromere proteins, CENPE is not detectedat centromeres during interphase, and first appears at the centromere region ofchromosomes during prometaphase. CENPE function is required for the transition frommetaphase to anaphase. CENPE is probably one of the motors responsible for mammalianchromosome movement and/or spindle elongation.

Characteristics: Synonyms: Centromere-associated protein E, Centromeric protein E, CENP-E, Kinesin-related proteinCENPE

Application Details

Protocol: Immunofluorescence protocol - Formaldehyde fixationCollect cells from T. c. unit and remove media from petri dish using suction. Wash with 1x PBS and remove. Incubate cells in pre-warm (37°C) Para-Formaldehyde for 12 minutes at room temperatureon an orbital shaker. Remove PFA and incubate in 0. 5% Triton X-IOO in 1x PBS for 5 minutes at roomtemperature. Prepare blocking reagent, this is also the antibody diluent. Wash cells 2x with 1x PBS at room temperature, for 4 minutes/wash on an orbital shaker. Block with 1 % NCS and 1x PBS for 30 minutes at room temperature. Prepare primary antibodies (50μl/coverslip) and moist staining chambers. Wash cells 2x with lx PBS at room temperature and air dry briefly. Incubate with primary antibody for 1 hr at room temperature in the dark in stainingchambers. During this time prepare the secondary antibody. Wash cells 5x with 1x PBS (5 beaker changes/5 counts in each beaker)Incubate with secondary antibody for 1 hour at room temperature in the dark in stainingchambers. Wash cells 5x with 1x PBS. Mount in Dapi. Solutions (prepare fresh the same day of staining). 1x Phosphate buffered saline. Blocking reagent: 1% NCS in 1x PBS (use fresh l0x PBS). Fixation solution: 3. 5% Para formaldehyde. 1. 75g PFA in 20 ml d. H20 plus 5 drops 1M NaOH. Stir on a hot plate at 50-60°C untildissolved. Add 4 drops IN HCI and check pH indicator strip. PH should be 7. 4. Completevolume with d. H20 to 25ml and add 25ml 2xPBS. Check pH before adding to cover slips. Immunofluorescence protocol - Methanol/acetone fixationCollect cells from T. C. unit and remove media from petri dish using suction. Wash with 1x PBS and remove. Fix cells with cold methanol: acetone 1: 1 for 10 minutes on ice. Prepare blocking reagent, this is also the diluent for the antibodies. Remove fixative and wash cells 3x with Ix PBS at RT, for 4 minutes/wash on orbital shaker. Block with 1% NCS and Ix PBS for 30 minutes at RT. Prepare primary antibodies (50μl/coverslip) and moist staining chambers. Wash cells 2x with 1 x PBS at RT and air dry for approximately 7 minutes. Incubate with primary antibody for 1 hr at RT in the dark in staining chambers. During thistime prepare secondary antibody. Wash cells 5x with 1x PBS (5 beaker changes/5 counts in each beaker)Incubate with secondary antibody for 1 hr at R T in the dark in staining chambers. Wash cells 5x with 1x PBS. Mount in Dapi. Solutions (prepare fresh the same day of staining)1x Phosphate buffered saline. Blocking reagent: 1% NCS in 1x PBS (use fresh 10x PBS). Fixation solution: methanol: acetone 1: 1 ice cold. Western Blotting ProtocolTransfer gel to PDVF or nitrocellulose membranePlace membrane in plastic tray in blocking buffer for one hour with agitation

Application Notes: Immunoprecipitation: Use ~3 mg/ml of antibody for 300 µg of cell lysate. Immunocytochemistry: Use 0.5-1 µg/ml as a guideline. Western blot: The antigen is first concentrated by Immunoprecipitation. Any Human cell line should be used for a Positive Control. Other applications not tested. Optimal dilutions are dependent on conditions and should be determined by the user.

Concentration: 0.45 mg/ml

Purification: Purified

Buffer: PBS with 0.09% sodium azide as preservative.

Storage: Store the antibody at 2-8°C for one month or (in small aliquots) at -20°C for longer. Avoid repeated freezing and thawing. Shelf life: One year from despatch.

Research Area: Chromatin and Nuclear Signaling, Cell Cycle

Restrictions: For Research Use only

Publications

Yen, Li, Schaar et al.: "CENP-E is a putative kinetochore motor that accumulates just before mitosis." in: Nature, Vol. 359, Issue 6395, pp. 536-9, 1992 (PubMed).

Yen, Compton, Wise et al.: "CENP-E, a novel human centromere-associated protein required for progression from metaphase to anaphase." in: The EMBO journal, Vol. 10, Issue 5, pp. 1245-54, 1991 (PubMed).