Anti-NAK/TBK1 antibody [EP611Y]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-NAK/TBK1 antibody [EP611Y] (AB40676)

ab40676 was shown to react with TBK1 in wild-type U2OSn cells in Immunocytochemistry with loss of signal observed in a TBK1 knockout cell line. Wild-type and Knockout cells were mixed and pelleted at a 1 : 1 ratio on coverslips. The cells were fixed with 4% paraformaldehyde (15 min) then permeabilized with 0.1% Triton X-100 (10min) and then blocked with 5%BSA+5%goat serum (30min). The cells were then incubated with ab40676 at 1/1500 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat anti-rabbit secondary antibody to (Alexa Fluor^® 555) at 0.5 μg/ml. Acquisition of the green (wild-type), red (antibody staining) and far-red (knockout) channels was performed. Representative grayscale images of the red channel are shown. Wild-type and knockout cells are outlined with yellow and magenta dashed line, respectively. Schematic representation of the mosaic strategy used is shown on the bottom-right panel. Image was acquired with a Zeiss(LSM-880).

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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-NAK/TBK1 antibody [EP611Y] (AB40676)

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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NAK/TBK1 antibody [EP611Y] (AB40676)

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.

• WB

Collaborator

Western blot - 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.

All lanes:

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

Predicted band size: 84 kDa

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• WB

Lab

Western blot - Anti-NAK/TBK1 antibody [EP611Y] (AB40676)

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.

All lanes:

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

Predicted band size: 84 kDa

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• WB

Unknown

Western blot - Anti-NAK/TBK1 antibody [EP611Y] (AB40676)

Blocking/Diluting buffer 5% NFDM/TBST

All lanes:

Western blot - Anti-NAK/TBK1 antibody [EP611Y] (ab40676) at 1/5000 dilution

Lane 1:

HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate at 20 µg

Lane 2:

SH-SY5Y (Human neuroblastoma cell line from bone marrow) whole cell lysate at 20 µg

Lane 3:

C6 (Rat glial tumor cell line) whole cell lysate at 20 µg

Lane 4:

NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 84 kDa

Observed band size: 84 kDa

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