Recombinant Anti-NAK/TBK1 antibody [EP611Y] (ab40676)

ab40676 was shown to react with TBK1 in wild-type U2OSn cells in Western blot with loss of signal observed in a TBK1 knockout cell line. Wild-type U2OSn and TBK1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab40676 overnight at 4 °C at a 1/10000 dilution. Blots were incubated with goat anti-rabbit HRP secondary antibodies at 1/5000 before imaging. These data were provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: NAK knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: HepG2 cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab40676 observed at 90 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab40676 was shown to specifically react with NAK when NAK knockout samples were used. Wild-type and NAK knockout samples were subjected to SDS-PAGE. ab40676 and ab8245 (loading control to GAPDH) were diluted at 1/1000 and 1/10,000  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.

Immunohistochemical analysis of paraffin-embedded human hepatocellular carcinoma tissue sections labeling NAK/TBK1 with purified ab40676 at a dilution of 1/100 (11.5 μg/ml). ab97051 Goat Anti-Rabbit IgG H&L (HRP) at 1/500 was used as the secondary anitbody. Sections were counterstained with hematoxylin. Antigen retrieval was heat mediated using EDTA Buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control and is shown in the inset. 

Immunocytochemistry/Immunofluorescence analysis of MCF7 (Human breast adenocarcinoma) cells labeling NAK/TBK1 with purified ab40676 at 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, Alexa Fluo^®488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. Cells were counter-stained with ab195889 Anti-Alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^®594) at 1/200. DAPI (blue) was used as a nuclear counterstain. Secondary Only Control: PBS was used instead of the primary antibody as the negative control.