Anti-Chk2 antibody [EPR19482]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Chk2 antibody [EPR19482] (AB207446)

Immunohistochemical analysis of paraffin-embedded Human testis tissue labeling Chk2 with ab207446 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on human spermatogonium is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Chk2 antibody [EPR19482] (AB207446)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Chk2 with ab207446 at 1/500 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining on HeLa cell line. The nuclear counter stain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution (red).

The negative controls are as follows :

-ve control 1 : ab207446 at 1/500 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary at 1/1000 dilution.

-ve control 2 : Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary at 1/1000 dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Chk2 antibody [EPR19482] (AB207446)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed HCT 116 (Human colorectal carcinoma cell line) cells labeling Chk2 with ab207446 at 1/70 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (Alexa Fluorr^® 488) at 1/500 dilution was used as the secondary antibody.

Note : Cells were permeabilised with 90% methanol-PBS, -20°C, 30min

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Chk2 antibody [EPR19482] (AB207446)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HCT 116 (Human colorectal carcinoma cell line) cells labeling Chk2 with ab207446 at 1/500 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining on HCT 116 cell line. The nuclear counter stain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution (red).
The negative controls are as follows :
-ve control 1 : ab207446 at 1/500 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary at 1/1000 dilution.
-ve control 2 : Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary at 1/1000 dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Chk2 antibody [EPR19482] (AB207446)

Immunohistochemical analysis of paraffin-embedded human breast cancer tissue labeling Chk2 with ab207446 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on tumor cells of human breast cancer is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IP

Supplier Data

Immunoprecipitation - Anti-Chk2 antibody [EPR19482] (AB207446)

Chk2 was immunoprecipitated from 1mg of HEK-293T (Human epithelial cell line from embryonic kidney) whole cell lysate with ab207446 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab207446 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : HEK-293T whole cell lysate 10μg (Input).

Lane 2 : ab207446 IP in HEK-293T whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab207446 in HEK-293T whole cell lysate.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 10 seconds.

All lanes:

Immunoprecipitation - Anti-Chk2 antibody [EPR19482] (ab207446)

Predicted band size: 61 kDa

false

• IP

Supplier Data

Immunoprecipitation - Anti-Chk2 antibody [EPR19482] (AB207446)

Chk2 was immunoprecipitated from 1mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab207446 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab207446 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : HeLa whole cell lysate 10μg (Input).

Lane 2 : ab207446 IP in HeLa whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab207446 in HeLa whole cell lysate.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 10 seconds.

All lanes:

Immunoprecipitation - Anti-Chk2 antibody [EPR19482] (ab207446)

Predicted band size: 61 kDa

false

• WB

Supplier Data

Western blot - Anti-Chk2 antibody [EPR19482] (AB207446)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1/3 : 15 seconds; Lane 2 : 8 seconds; Lane 4 : 30 seconds; Lane 5 : 3 minutes.

Lanes 1, 2, 3 and 5:

Western blot - Anti-Chk2 antibody [EPR19482] (ab207446) at 1/1000 dilution

Lane 4:

Western blot - Anti-Chk2 antibody [EPR19482] (ab207446) at 1/5000 dilution

Lane 1:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 10 µg

Lane 2:

HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 10 µg

Lane 3:

HEK-293T (Human epithelial cell line from embryonic kidney) whole cell lysate at 10 µg

Lane 4:

HCT 116 (Human colorectal carcinoma cell line) whole cell lysate at 10 µg

Lane 5:

SH-SY5Y (Human neuroblastoma cell line from bone marrow) whole cell lysate at 10 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 1/100000 dilution

Predicted band size: 61 kDa

Observed band size: 61 kDa

false

• WB

Lab

Western blot - Anti-Chk2 antibody [EPR19482] (AB207446)

False colour image of Western blot : Anti-Chk2 antibody [EPR19482] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab207446 was shown to bind specifically to Chk2. A band was observed at 67 kDa in wild-type A549 cell lysates with no signal observed at this size in CHEK2 knockout cell line ab276098 (knockout cell lysate ab276098). To generate this image, wild-type and CHEK2 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

Lanes 1 - 4:

Western blot - Anti-Chk2 antibody [EPR19482] (ab207446) at 1/1000 dilution

Lanes 1 - 4:

Western blot - Anti-Chk2 antibody [EPR19482] - BSA and Azide free (<a href='/en-us/products/primary-antibodies/chk2-antibody-epr19482-bsa-and-azide-free-ab251477'>ab251477</a>) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

Western blot - Human CHEK2 (Chk2) knockout A549 cell line (<a href='/en-us/products/cell-lines/human-chek2-chk2-knockout-a549-cell-line-ab276098'>ab276098</a>)

Lane 2:

CHEK2 knockout A549 cell lysate at 20 µg

Lane 3:

HEK-293 cell lysate at 20 µg

Lane 4:

MDA-MB-231 cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 67 kDa

false

• WB

Supplier Data

Western blot - Anti-Chk2 antibody [EPR19482] (AB207446)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Chk2 antibody [EPR19482] (ab207446) at 1/1000 dilution

Lane 1:

Human colon lysate at 10 µg

Lane 2:

Human thymus lysate at 10 µg

Lane 3:

Human kidney lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 61 kDa

Observed band size: 61 kDa

false

Exposure time: 3min

• WB

Lab

Western blot - Anti-Chk2 antibody [EPR19482] (AB207446)

Lanes 1-4 : Merged signal (red and green). Green - ab207446 observed at 68 kDa. Red - loading control ab8245 observed at 37 kDa.

ab207446 Anti-Chk2 antibody [EPR19482] was shown to specifically react with Chk2 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab264815 (knockout cell lysate ab257104) was used. Wild-type and Chk2 knockout samples were subjected to SDS-PAGE. ab207446 and Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker (ab52866) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. 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 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Chk2 antibody [EPR19482] (ab207446) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

CHEK2 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human CHEK2 (Chk2) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-chek2-chk2-knockout-hela-cell-line-ab264815'>ab264815</a>)

Lane 3:

HEK-293 cell lysate at 20 µg

Lane 4:

MDA-MB-231 cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution

Predicted band size: 61 kDa

Observed band size: 68 kDa

false

• WB

Lab

Western blot - Anti-Chk2 antibody [EPR19482] (AB207446)

Lanes 1 - 3 : Merged signal (red and green). Green - ab207446 observed at 68 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab207446 was shown to specifically recognize CHEK2 in wild-type HAP1 cells along with additional cross-reactive bands. No band was observed when CHEK2 knockout samples were examined. Wild-type and CHEK2 knockout samples were subjected to SDS-PAGE. ab207446 and ab9484 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Chk2 antibody [EPR19482] (ab207446) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

CHEK2 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HeLa whole cell lysate at 20 µg

Predicted band size: 61 kDa

false