The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 µg/ml.
Use at an assay dependent dilution.
1/500 - 1/1000. Detects a band of approximately 180 kDa (predicted molecular weight: 160 kDa).
Multifunctional enzyme that has both magnesium and ATP-dependent DNA-helicase activity and 3'->5' exonuclease activity towards double-stranded DNA with a 5'-overhang. Has no nuclease activity towards single-stranded DNA or blunt-ended double-stranded DNA. Binds preferentially to DNA substrates containing alternate secondary structures, such as replication forks and Holliday junctions. May play an important role in the dissociation of joint DNA molecules that can arise as products of homologous recombination, at stalled replication forks or during DNA repair. Alleviates stalling of DNA polymerases at the site of DNA lesions. Important for genomic integrity. Plays a role in the formation of DNA replication focal centers; stably associates with foci elements generating binding sites for RP-A.
Involvement in disease
Defects in WRN are a cause of Werner syndrome (WRN) [MIM:277700]. WRN is a rare autosomal recessive progeroid syndrome characterized by the premature onset of multiple age-related disorders, including atherosclerosis, cancer, non-insulin-dependent diabetes mellitus, ocular cataracts and osteoporosis. The major cause of death, at a median age of 47, is myocardial infarction. Currently all known WS mutations produces prematurely terminated proteins. Defects in WRN may be a cause of colorectal cancer (CRC) [MIM:114500].
Western blot - Anti-Werner's syndrome helicase WRN antibody [8H3] (ab66601)
Predicted band size : 160 kDa
Lane 1: Wild-type HAP1 cell lysate (20 µg) Lane 2: Werner's syndrome helicase WRN knockout HAP1 cell lysate (20 µg) Lane 3: MOLT4 cell lysate (20 µg) Lane 4: K562 cell lysate (20 µg) Lanes 1 to 4: Merged signal (red and green). Green - ab66601 observed at 170 kDa. Red - loading control, ab181602, observed at 37 kDa. ab66601 was shown to recognize Werner's syndrome helicase WRN when Werner's syndrome helicase WRN knockout samples were used, along with additional cross-reactive bands. Wild-type and Werner's syndrome helicase WRN knockout samples were subjected to SDS-PAGE. ab66601 and ab181602 (loading control to GAPDH) were both diluted at 1/500 and 1/10000 and incubated overnight at 4°C. Blots were developed with Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed ab216772 and Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed ab216777 secondary antibodies at 1/10000 dilution for 1 h at room temperature before imaging.
WRN helicase is illustrated above.The helicase domain is shown in black, and the nuclear localizing signal region is hatched. Epitope sites of 4H12 and 8H3 identified by the studies with various recombinant WRN helicases (Shiratori et. al., J. Cell Biol. 144: 1-9 (1999) are also shown.WRN helicase is illustrated above.The helicase domain is shown in black, and the nuclear localizing signal region is hatched. Epitope sites of 4H12 and 8H3 identified by the studies with various recombinant WRN helicases (Shiratori et. al., J. Cell Biol. 144: 1-9 (1999) are also shown.
Western blot - Werner's syndrome helicase WRN antibody [8H3] - Aminoterminal end (ab66601)
All lanes : Anti-Werner's syndrome helicase WRN antibody [8H3] (ab66601) at 1/500 dilution
Lane 1 : Extracts from cells of normal individuals Lane 2 : Extracts from cells of normal individuals Lane 3 : Extracts from cells of normal individuals Lane 4 : Extracts of cells from patients with Werner syndrome Lane 5 : Extracts of cells from patients with Werner syndrome Lane 6 : Purified recombinant WRN helicase made by baculovirus technology in the insect SF9 cells. See details published in the paper by Goto et al., Human Genet 105:301-307 (1999).
Predicted band size : 160 kDa Western blot analysis by ab66601 of WRN helicase protein in the EBV-transformed cells from WS (Werner syndrome) patient cells and normal individuals.
ICC/IF image of ab66601 stained HeLa cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab66601, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.