All lanes : Anti-Chk2 antibody [EPR4325] (ab109413) 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 - ab109413 observed at 68 kDa. Red - loading control ab8245 observed at 37 kDa.

 ab109413 Anti-Chk2 antibody [EPR4325] 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. ab109413 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.

 

Lanes 1-4 : Anti-Chk2 antibody [EPR4325] (ab109413) at 1/50000 dilution
Lanes 5-8 : Anti-GAPDH antibody [6C5] - Loading Control (ab8245) at 1/2000 dilution

Lanes 1 & 5 : Wild-type HAP1 cell lysate
Lanes 2 & 6 : Chk2 knockout HAP1 cell lysate
Lanes 3 & 7 : HeLa cell lysate
Lanes 4 & 8 : HEK293 cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 61 kDa

Lanes 1, 2, 3 and 4: Green signal from target -  ab109413 observed at 62 kDa
Lanes 5, 6, 7 and 8: Red signal from loading control - ab8245 observed at 37 kDa
Lanes 9, 10, 11 and 12: Merged (red and green) signal

ab109413 was shown to specifically react with Chk2 when Chk2 knockout samples were used. Wild-type and Chk2 knockout samples were subjected to SDS-PAGE. ab109413 and ab8245 (loading control to GAPDH) were diluted 1/50 000 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.

ab109413 staining Chk2 in wild-type HAP1 cells (top panel) and Chk2 knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5 minutes), 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 1 hour. The cells were then incubated with ab109413 at 1/250 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 1 hour 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.

Immunohistochemical analysis of paraffin-embedded human colon tissue using ab109413 at a 1/100 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling Chk2 with purified ab109413 at 1/230 dilution(10 µg/ml) (red). Cells were fixed with 80% methanol and permeabilised with 0.1% Tween-20. A Goat anti rabbit IgG (Alexa Fluor^® 488) (ab150077) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black)(ab172730) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control.

Purified ab109413 at 1/50 dilution (2µg) immunoprecipitating Chk2 in HeLa whole cell lysate.
Lane 1 (input): HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10µg
Lane 2 (+): ab109413 + HeLa whole cell lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab109413 in HeLa whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM/TBST.
Observed band size: 62 kDa

All lanes : Anti-Chk2 antibody [EPR4325] (ab109413)

Lane 1 : Wild-type HAP1 cell lysate
Lane 2 : Chk2 knockout HAP1 cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : HEK293 cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 61 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab109413 observed at 64 kDa. Red - loading control, ab8245, observed at 37 kDa.

This western blot image is a comparison between ab109413 and a competitor's rabbit polyclonal antibody.

 

Immunocytochemistry analysis of HT-29 (human colorectal adenocarcinoma epithelial cell) labeling Chk2 with purified ab109413 at 1/500 dilution. Cells were fixed with 4% Paraformaldehyde and permeabilised with 0.1% tritonX-100. Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/1000 (2 µg/ml) was used as the secondary antibody. was used as counterstain. Nuclei were stained blue with DAPI.
Negative control: PBS instead of the primary antibody.

All lanes : Anti-Chk2 antibody [EPR4325] (ab109413) at 1/50000 dilution

Lane 1 : HeLa (human epithelial cell line from cervix adenocarcinoma) cells treated with gamma irradiation
Lane 2 : HeLa (human epithelial cell line from cervix adenocarcinoma) cell lysate
Lane 3 : HT-29 (human colorectal adenocarcinoma cell line) cell lysate
Lane 4 : HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) cell lysate

Lysates/proteins at 10 µg per lane.

Predicted band size: 61 kDa
Observed band size: 62 kDa why is the actual band size different from the predicted?

Immunohistochemical analysis of paraffin-embedded human spleen tissue using ab109413 at a 1/100 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

ab109413 (1/500) staining Chk2 in HeLa (human epithelial cell line from cervix adenocarcinoma) cells (green). Cells were fixed in paraformaldehyde, permeabilized with 0.5% Triton X-100/PBS and counterstained with DAPI in order to highlight the nucleus (red). For further experimental details please see Abreview.

See Abreview