The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/1000 - 1/10000. Detects a band of approximately 75 kDa (predicted molecular weight: 67 kDa).
1/50 - 1/100. Perform heat mediated antigen retrieval via the microwave method before commencing with IHC staining protocol. (Heat to 98°C, allow to cool for 10-20 minutes)
Use at an assay dependent concentration.
Application notesIs unsuitable for IP.
FunctionComponent of a protein kinase signal transduction cascade. Mediator of TRAF6 and TGF-beta signal transduction. Activates IKBKB and MAPK8 in response to TRAF6 signaling. Stimulates NF-kappa-B activation and the p38 MAPK pathway. In osmotic stress signaling, plays a major role in the activation of MAPK8/JNK, but not that of NF-kappa-B.
Sequence similaritiesBelongs to the protein kinase superfamily. STE Ser/Thr protein kinase family. MAP kinase kinase kinase subfamily. Contains 1 protein kinase domain.
Post-translational modificationsAssociation with TAB1/MAP3K7IP1 promotes autophosphorylation and subsequent activation. Association with TAB2/MAP3K7IP2, itself associated with free unanchored Lys-63 polyubiquitin chain, promotes autophosphorylation and subsequent activation of MAP3K7. Dephosphorylation at Thr-187 by PP2A and PPP6C leads to inactivation. Ubiquitinated, leading to proteasomal degradation (By similarity). Requires 'Lys-63'-linked polyubiquitination for autophosphorylation and subsequent activation. 'Lys-63'-linked ubiquitination does not lead to proteasomal degradation. Deubiquitinated by CYLD, a protease that selectively cleaves 'Lys-63'-linked ubiquitin chains. Deubiquitinated by Y.enterocolitica YopP.
Western blot - Anti-TAK1 antibody [EPR5984] (ab109526)
Predicted band size : 67 kDa
Lane 1: Wild-type HAP1 cell lysate (20 µg) Lane 2: TAK1 knockout HAP1 cell lysate (20 µg) Lane 3: SHSY5Y cell lysate (20 µg) Lane 4: A431 cell lysate (20 µg) Lanes 1 - 4: Merged signal (red and green). Green - ab109526 observed at 72 kDa. Red - loading control, ab8245, observed at 37 kDa. ab109526 was shown to specifically react with TAK1 when TAK1 knockout samples were used. Wild-type and TAK1 knockout samples were subjected to SDS-PAGE. ab109526 and ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/2000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging.
ab109526 staining TAK1 in wild-type HAP1 cells (top panel) and TAK1 knockout HAP1 cells (bottom panel). The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab109526 at 1/1000 dilution and ab195889 at 1/250 dilution (shown in pseudo colour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
Flow Cytometry analysis of A431(human epidermoid carcinoma) cells labeling TAK1 with unpurified ab109526 at 1/20 dilution (10ug/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor® 488)(1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) was used as the unlabeled control.
Western blot - TAK1 antibody [EPR5984] (ab109526)
All lanes : Anti-TAK1 antibody [EPR5984] (ab109526) at 1/1000 dilution
Lane 1 : K562 cell lysate Lane 2 : HeLa cell lysate Lane 3 : A431 cell lysate
Lysates/proteins at 10 µg per lane.
Secondary HRP labelled Goat anti-Rabbit IgG at 1/2000 dilution