重组Anti-Chk2抗体[EPR4325]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Chk2 antibody [EPR4325] (AB109413)

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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Chk2 antibody [EPR4325] (AB109413)

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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Chk2 antibody [EPR4325] (AB109413)

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.

Image courtesy of an Abreview submitted by Dr. Kirk McManus, Univ. of Manitoba/Cancer Care MICB, Canada

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Chk2 antibody [EPR4325] (AB109413)

Intracellular Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling Chk2 with purified ab109413 at 1/230 dilution (10μg/ml) (red). Cells were fixed with 80% methanol and permeabilised with 0.1% Tween-20. A Goat anti rabbit IgG (Alexa Fluorr^® 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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Chk2 antibody [EPR4325] (AB109413)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Chk2 antibody [EPR4325] (AB109413)

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.

• IP

Lab

Immunoprecipitation - Anti-Chk2 antibody [EPR4325] (AB109413)

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:

Immunoprecipitation - Anti-Chk2 antibody [EPR4325] (ab109413)

Predicted band size: 61 kDa

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• WB

Lab

Western blot - Anti-Chk2 antibody [EPR4325] (AB109413)

False colour image of Western blot : Anti-Chk2 antibody [EPR4325] staining at 1/50000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab109413 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 [EPR4325] (ab109413) at 1/5000 dilution

Lanes 1 - 4:

Western blot - Anti-Chk2 antibody [EPR4325] - BSA and Azide free (<a href='/products/primary-antibodies/chk2-antibody-epr4325-bsa-and-azide-free-ab227998'>ab227998</a>) at 1/5000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

Western blot - Human CHEK2 (Chk2) knockout A549 cell line (<a href='/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

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• WB

Lab

Western blot - Anti-Chk2 antibody [EPR4325] (AB109413)

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.

All lanes:

Western blot - Anti-Chk2 antibody [EPR4325] (ab109413) 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='/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='/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

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• WB

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Western blot - Anti-Chk2 antibody [EPR4325] (AB109413)

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.

Lanes 1 - 4:

Western blot - Anti-Chk2 antibody [EPR4325] (ab109413) at 1/50000 dilution

Lanes 5 - 8:

Western blot - Anti-Chk2 antibody [EPR4325] (ab109413) at 1/2000 dilution

Lanes 1 and 5:

Wild-type HAP1 cell lysate at 20 µg

Lanes 2 and 6:

Chk2 knockout HAP1 cell lysate at 20 µg

Lanes 3 and 7:

HeLa cell lysate at 20 µg

Lanes 4 and 8:

HEK293 cell lysate at 20 µg

Predicted band size: 61 kDa

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• WB

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Western blot - Anti-Chk2 antibody [EPR4325] (AB109413)

All lanes:

Western blot - Anti-Chk2 antibody [EPR4325] (ab109413) at 1/50000 dilution

Lane 1:

HeLa (human epithelial cell line from cervix adenocarcinoma) cells treated with gamma irradiation at 10 µg

Lane 2:

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

Lane 3:

HT-29 (human colorectal adenocarcinoma cell line) cell lysate at 10 µg

Lane 4:

HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) cell lysate at 10 µg

Predicted band size: 61 kDa

Observed band size: 62 kDa

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• WB

Lab

Western blot - Anti-Chk2 antibody [EPR4325] (AB109413)

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.

All lanes:

Western blot - Anti-Chk2 antibody [EPR4325] (ab109413)

Lane 1:

Wild-type HAP1 cell lysate at 20 µg

Lane 2:

Chk2 knockout HAP1 cell lysate at 20 µg

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

HEK293 cell lysate at 20 µg

Predicted band size: 61 kDa

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