Anti-Chk1 antibody [EP691Y] - BSA and Azide free

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Chk1 antibody [EP691Y] - BSA and Azide free (AB210964)

Clone EP691Y (ab210964) has been successfully conjugated by Abcam. This image was generated using Anti-Chk1 antibody [EP691Y] (Alexa Fluor® 647). Please refer to ab196520 for protocol details.

ab196520 staining Chk1 in MCF7 cells. The cells were fixed with 4% formaldehyde (10 min), permeabilized in 0.1% Triton X-100 for 5 minutes and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab196520 at 1/100 dilution (shown in red) and ab195887, Mouse monoclonal [DM1A] to alpha Tubulin (Alexa Fluor^® 488, shown in green) at 1/167 dilution, overnight at +4°C. Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Chk1 antibody [EP691Y] - BSA and Azide free (AB210964)

Clone EP691Y (ab210964) has been successfully conjugated by Abcam. This image was generated using Anti-Chk1 antibody [EP691Y] (Alexa Fluor® 488). Please refer to ab196308 for protocol details.

ab196308 staining Chk1 in MCF-7cells. The cells were fixed with 4% formaldehyde (10 min), permeabilised in 0.1% Triton X-100 for 5 minutes and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated with ab196308 at 1/500 dilution (shown in green) and ab195889, Mouse monoclonal [DM1A] to alpha Tubulin (Alexa Fluor^® 594, shown in red) at 1/167 dilution overnight at +4°C. Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Chk1 antibody [EP691Y] - BSA and Azide free (AB210964)

Immunohistochemical analysis of Chk1 expression in paraffin-embedded human breast carcinoma using ab40866 at a 1 : 250 dilution. Heat mediated antigen retrieval was performed with citrate buffer pH 6 before commencing with IHC staining protocol.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab40866).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Chk1 antibody [EP691Y] - BSA and Azide free (AB210964)

Immunofluorescence staining of MCF7 cells with purified ab40866 at a working dilution of 1/500, counter-stained with DAPI. The secondary antibody was an Alexa Fluor^® 488 conjugated goat anti-rabbit (ab150077), used at a dilution of 1/1000. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative control is shown in bottom right hand panel - for the negative control, PBS was used instead of the primary antibody.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab40866).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Chk1 antibody [EP691Y] - BSA and Azide free (AB210964)

Overlay histogram showing HeLa cells stained with ab40866 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab40866, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab40866).

• WB

Lab

Western blot - Anti-Chk1 antibody [EP691Y] - BSA and Azide free (AB210964)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab40866). Anti-CHEK1 antibody [EP691Y] (ab40866) staining at 1/10000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab40866 was shown to bind specifically to CHEK1. A band was observed at 57 kDa in wild-type A549 cell lysates with a reduction in signal observed at this size in CHEK1 heterozygous knockout cell line. To generate this image, wild-type and CHEK1 heterozygous 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.

All lanes:

Western blot - Anti-Chk1 antibody [EP691Y] (<a href='/en-us/products/primary-antibodies/chk1-antibody-ep691y-ab40866'>ab40866</a>) at 1/10000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

Western blot - Human CHEK1 heterozygous knockout A549 cell line (<a href='/en-us/products/cell-lines/human-chek1-heterozygous-knockout-a549-cell-line-ab276102'>ab276102</a>)

Lane 2:

CHEK1 knockout A549 cell lysate at 20 µg

Lane 3:

A431 cell lysate at 20 µg

Lane 4:

MOLT-4 cell lysate at 20 µg

Secondary

Lanes 1 - 4:

Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution

Lanes 1 - 4:

Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Predicted band size: 54 kDa

Observed band size: 57 kDa

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