Flap Structure-Specific Endonuclease 1 (FEN1) (AA 252-371) antibody

Antigen: Flap Structure-Specific Endonuclease 1 (FEN1)

Synonyms: MF1, RAD2, FEN-1, FEN1

Binding Site: AA 252-371

Clonality: Monoclonal (21)

Host: Mouse

Reactivity: Human

Conjugate: Un-conjugated

Application: Western Blotting (WB), BioImaging (BI)

Catalog no.: ABIN968459

Additional Information

Alternative name: FEN-1

Immunogen: Human FEN-1

Cross-Reactivity: Dog (Canine), Mouse (Murine), Rat (Rattus)

Format: Liquid

Isotype: IgG1

Clone: 21

Description: The fidelity of DNA replication, recombination, and repair is essential for genome stability. Proteins that have exonuclease and endonuclease enzymatic activity are critical for accurate replication, recombination, and repair of DNA sequences. FEN-1 ( five' exonuclease-1 or flap endonuclease-1) is an exo- and endonuclease found in yeast, mouse, and human. The 5'-3' exonuclease activity of FEN-1 involves only double stranded DNA and is important for processing Okazaki fragments during lagging strand DNA synthesis. In addition, FEN-1 is a member of the RAD2 family of repair nucleases and may participate in DNA repair. Its endonuclease activity is specific for 5' overhanging flaps, which are intermediates in the repair of DNA double strand breaks. Human and mouse FEN-1 are highly conserved with 95% identity at the amino acid level. FEN-1 expression is highest during entry into the mitotic cell cycle and lowest in differentiated cells. Thus, FEN-1 is an exo- and endonuclease that is thought to be essential for maintaining DNA integrity during replication, as well as after damage by alkylating agents or UV.

Characteristics: 1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
2. Please refer to us for technical protocols.
3. This antibody has been developed and certified for the bioimaging application. However, a routine bioimaging test is not performed on every lot. Researchers are encouraged to titrate the reagent for optimal performance.
4. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
5. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
6. Triton is a trademark of the Dow Chemical Company.

Molecular Weight: 50 kDa

Comments:

Related Products: ABIN967389, ABIN968536

Application Details

Application Notes: Bioimaging
1. Seed the cells in appropriate culture medium at ~10,000 cells per well in an 96-well Imaging Plate and culture overnight.
2. Remove the culture medium from the wells, and fix the cells by adding 100 myl of Fixation Buffer to each well. Incubate for 10 minutes at room temperature (RT).
3. Remove the fixative from the wells, and permeabilize the cells using either 90% methanol, or Triton™ X-100: a. Add 100 myl of -20°C 90% methanol to each well and incubate for 5 minutes at RT. OR b. Add 100 myl of 0.1% Triton™ X-100 to each well and incubate for 5 minutes at RT.
4. Remove the permeabilization buffer, and wash the wells twice with 100 myl of 1× PBS.
5. Remove the PBS, and block the cells by adding 100 myl of to each well. Incubate for 30 minutes at RT.
6. Remove the blocking buffer and add 50 myl of the optimally titrated primary antibody (diluted in Stain Buffer) to each well, and incubate for 1 hour at RT.
7. Remove the primary antibody, and wash the wells three times with 100 myl of 1× PBS.
8. Remove the PBS, and add the second step reagent at its optimally titrated concentration in 50 myl to each well, and incubate in the dark for 1 hour at RT.
9. Remove the second step reagent, and wash the wells three times with 100 myl of 1× PBS.
10. Remove the PBS, and counter-stain the nuclei by adding 200 myl per well of 2 myg/ml Hoechst 33342 in 1× PBS to each well at least 15 minutes before imaging.
11. View and analyze the cells on an appropriate imaging instrument.

Concentration: 250 µg/ml

Purity: Purified

Purification: Purified from tissue culture supernatant or ascites by affinity chromatography.

Buffer: Aqueous buffered solution containing BSA, glycerol.

Preservative: 0.09% Sodium azide.

Storage: Store undiluted at -20°C.

Restrictions: For Research Use only

Images

Western blot analysis of FEN-1 on a human endothelial lysate. Lane 1: 1:250, lane 2: 1:500, lane 3: 1:1000 dilution of the FEN-1 antibody.

Immunofluorescent staining of HeLa (ATCC CCL-2) cells. Cells were seeded in a 96 well imaging plate at ~ 10 000 cells per well. After overnight incubation, cells were stained using the Triton™ X-100 perm protocol and the anti-FEN-1 antibody. The second step reagent was FITC goat anti mouse Ig. Images were taken on a BD Pathway™ 855 Bioimager using a 20x objective. This antibody also stained A549 (ATCC CCL-185) and U-2 OS (ATCC HTB-96) cells and worked with both the Triton™ X-100 and alcohol perm protocols.

anti-Flap Structure-Specific Endonuclease 1 (FEN1) (AA 252-371) antibody (Image 3)

Publications

Product: Hiraoka, Harrington, Gerhard et al.: "Sequence of human FEN-1, a structure-specific endonuclease, and chromosomal localization of the gene (FEN1) in mouse and human." in: Genomics, Vol. 25, Issue 1, pp. 220-5, 1995 (PubMed).

Harrington, Lieber: "The characterization of a mammalian DNA structure-specific endonuclease." in: The EMBO journal, Vol. 13, Issue 5, pp. 1235-46, 1994 (PubMed).

Hosfield, Mol, Shen et al.: "Structure of the DNA repair and replication endonuclease and exonuclease FEN-1: coupling DNA and PCNA binding to FEN-1 activity." in: Cell, Vol. 95, Issue 1, pp. 135-46, 1998 (PubMed).

Kim: "Down-regulation of human FEN-1 gene expression during differentiation of promyelocytic leukemia cells." in: Experimental & molecular medicine, Vol. 30, Issue 4, pp. 252-6, 1999 (PubMed).

Yoon, Swiderski, Kaplan et al.: "Processing of UV damage in vitro by FEN-1 proteins as part of an alternative DNA excision repair pathway." in: Biochemistry, Vol. 38, Issue 15, pp. 4809-17, 1999 (PubMed).