Anti-Chk1 抗体 [EP691Y]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Chk1 antibody [EP691Y] (AB40866)

Immunohistochemical analysis of Chk1 expression in paraffin-embedded human breast carcinoma using ab40866 at a 1 : 250 dilution.

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

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Chk1 antibody [EP691Y] (AB40866)

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.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Chk1 antibody [EP691Y] (AB40866)

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.

• WB

Supplier Data

Western blot - Anti-Chk1 antibody [EP691Y] (AB40866)

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

UV-C treatment induces phosphorylation of Chk1 at Ser345 (PMID : 20609246).

The identity of the bands higher than 150 kDa are unknown.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

In Western blot, Anti-Chk1 antibody [EP691Y] (ab40866) staining at 56KDa dilution.

All lanes:

Western blot - Anti-Chk1 (phospho S345) antibody [EPR30270-566] (<a href='/products/primary-antibodies/chk1-phospho-s345-antibody-epr30270-566-ab325675'>ab325675</a>) at 1/1000 dilution

Lane 1:

Untreated Synchronized G1/S HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate (untreated membrane) at 20 µg

Lane 2:

Synchronized G1/S HeLa treated with 100 J/m2 UV-C, then recovery for 1 hour whole cell lysate (untreated membrane) at 20 µg

Lane 3:

Untreated Synchronized G1/S HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate (phosphatase treated membrane) at 20 µg

Lane 4:

Synchronized G1/S HeLa treated with 100 J/m2 UV-C, then recovery for 1 hour whole cell lysate (phosphatase treated membrane) at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 56 kDa,36 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-Chk1 antibody [EP691Y] (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] (ab40866) 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='/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

false

• WB

Unknown

Western blot - Anti-Chk1 antibody [EP691Y] (AB40866)

All lanes:

Western blot - Anti-Chk1 antibody [EP691Y] (ab40866) at 1/10000 dilution

All lanes:

HeLa cell lysate at 10 µg

Predicted band size: 54 kDa

Observed band size: 54 kDa

false