Anti-Chk1 (phospho S345) antibody [EPR30270-566]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Chk1 (phospho S345) antibody [EPR30270-566] (AB325675)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Synchronized G1/S HeLa (human cervical adenocarcinoma epithelial cell), Synchronized G1/S HeLa cells treated with UV-C (100 J/m2) and then recovery for 1 hr and Synchronized G1/S HeLa cells treated with UV-C (100 J/m2) and then recovery for 1 hr and treated with phosphatase at 37℃ for 2h labelling Chk1 (phospho S345) with ab325675 at 1/500 (1.02 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/ml) dilution (Green).

Confocal image showing increased nuclear staining in HeLa cells (shown in green) treated with UV-C (100 J/m2) and then recovery for 1 hr, and the signal decreased after phosphatase treatment at 37℃ for 2h. The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 (1 ug/ml) dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 (2 ug/ml) dilution (Magenta).

• WB

Supplier Data

Western blot - Anti-Chk1 (phospho S345) antibody [EPR30270-566] (AB325675)

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] (ab325675) 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='/en-us/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

Supplier Data

Western blot - Anti-Chk1 (phospho S345) antibody [EPR30270-566] (AB325675)

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

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

In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) staining at 1/5000 dilution.

All lanes:

Western blot - Anti-Chk1 (phospho S345) antibody [EPR30270-566] (ab325675) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) transfected with an empty vector containing a myc-His-tag® whole cell lysate at 20 µg

Lane 2:

293T transfected with a human wild-type Chk1 expression vector containing a myc-His-tag® whole cell lysate at 20 µg

Lane 3:

293T transfected with a human Chk1 (S345A mutation) expression vector containing a myc-His-tag® whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/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: 37s

• Dot

Supplier Data

Dot Blot - Anti-Chk1 (phospho S345) antibody [EPR30270-566] (AB325675)

Dot blot analysis of Chk1 (phospho S345) using ab325675 at 1 : 1000 (0.51 ug/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1 : 100,000 dilution.

Lane1 : Human Chk1 (phospho S345) peptide a
Lane2 : Human Chk1 (phospho S345) peptide b
Lane3 : Human Chk1 (non-phospho) peptide

Exposure time : 180 seconds.

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

All lanes:

Dot Blot - Anti-Chk1 (phospho S345) antibody [EPR30270-566] (ab325675) at 1/1000 dilution

Lane 1:

Human Chk1 (phospho S345) peptide a

Lane 2:

Human Chk1 (phospho S345) peptide b

Lane 3:

Human Chk1 (non-phospho) peptide

Secondary

All lanes:

Dot Blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

false

Exposure time: 180s