All lanes : Anti-Chk2 antibody [EPR19482] (ab207446) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : CHEK2 knockout HeLa cell lysate
Lane 3 : HEK-293 cell lysate
Lane 4 : MDA-MB-231 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 61 kDa
Observed band size: 68 kDa why is the actual band size different from the predicted?

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

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : CHEK2 knockout HAP1 whole cell lysate
Lane 3 : HeLa whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 61 kDa

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.

Lanes 1-3 & 5 : Anti-Chk2 antibody [EPR19482] (ab207446) at 1/1000 dilution
Lane 4 : Anti-Chk2 antibody [EPR19482] (ab207446) at 1/5000 dilution

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate
Lane 3 : HEK-293T (Human epithelial cell line from embryonic kidney) whole cell lysate
Lane 4 : HCT 116 (Human colorectal carcinoma cell line) whole cell lysate
Lane 5 : SH-SY5Y (Human neuroblastoma cell line from bone marrow) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 61 kDa
Observed band size: 61 kDa

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.

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.

All lanes : Anti-Chk2 antibody [EPR19482] (ab207446) at 1/1000 dilution

Lane 1 : Human colon lysate
Lane 2 : Human thymus lysate
Lane 3 : Human kidney lysate

Lysates/proteins at 10 µg per lane.

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


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

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.

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.

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.

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 Fluor^® 488) at 1/500 dilution was used as the secondary antibody.

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

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.

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.