Anti-RPA32/RPA2 (phospho S33) antibody [EPR29612-551]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-RPA32/RPA2 (phospho S33) antibody [EPR29612-551] (AB325750)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa (human cervical adenocarcinoma epithelial cell), HeLa treated with Etoposide(100uM) for 16hours and HeLa treated with Etoposide(100uM) for 16hours then Alkaline Phosphatase treatment 37℃ for 1 hour cells labelling RPA32/RPA2 (phospho S33) with ab325750 at 1/250 (2.06 μg/ml) dilution , followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing nuclear staining in HeLa cells treated with Etoposide(100uM) for 16hours and weak staining in HeLa cells and HeLa cells treated with Etoposide(100uM) for 16hours then Alkaline Phosphatase treatment 37℃ for 1 hour(shown in green). 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).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-RPA32/RPA2 (phospho S33) antibody [EPR29612-551] (AB325750)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast), NIH/3T3 treated with Camptothecin/CPT(1uM) for 3hours and NIH/3T3 treated with Camptothecin/CPT(1uM) for 3hours then Alkaline Phosphatase treatment 37℃ for 1 hour cells labelling RPA32/RPA2 (phospho S33) with ab325750 at 1/250 (2.06 μg/ml) dilution , followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing nuclear staining in NIH/3T3 cells treated with Camptothecin/CPT(1uM) for 3hours and weak staining in NIH/3T3 cells and NIH/3T3 cells treated with Camptothecin/CPT(1uM) for 3hours then Alkaline Phosphatase treatment 37℃ for 1 hour(shown in green). 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

Lab

Western blot - Anti-RPA32/RPA2 (phospho S33) antibody [EPR29612-551] (AB325750)

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

The molecular weight of RPA32/RPA2 is upshifted in response to Etoposide treatment (PMID : 17928296).

To minimize protein degradation, cells were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-RPA32/RPA2 (phospho S33) antibody [EPR29612-551] (ab325750) at 1/1000 dilution

Lane 1:

Untreated HeLa (human cervical adenocarcinoma epithelial cell) fresh whole cell lysate at 50 µg

Lane 2:

HeLa treated with 100 μM Etoposide for 16 hours fresh whole cell lysate at 50 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: 32 kDa,36 kDa,30 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-RPA32/RPA2 (phospho S33) antibody [EPR29612-551] (AB325750)

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

The molecular weight of RPA32/RPA2 is upshifted in response to camptothecin treatment (PMID : 17928296).

To minimize protein degradation, cells were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-RPA32/RPA2 (phospho S33) antibody [EPR29612-551] (ab325750) at 1/1000 dilution

Lane 1:

Untreated 293T (human embryonic kidney epithelial cell) fresh whole cell lysate (untreated membrane) at 50 µg

Lane 2:

293T treated with 1 μM camptothecin for 3 h fresh whole cell lysate (untreated membrane) at 50 µg

Lane 3:

Untreated 293T (human embryonic kidney epithelial cell) fresh whole cell lysate (alkaline phosphatase treated membrane) at 50 µg

Lane 4:

293T treated with 1 μM camptothecin for 3 h fresh whole cell lysate (alkaline phosphatase treated membrane) at 50 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: 32 kDa,36 kDa,30 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-RPA32/RPA2 (phospho S33) antibody [EPR29612-551] (AB325750)

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

The molecular weight of RPA32/RPA2 is upshifted in response to camptothecin treatment (PMID : 17928296).

To minimize protein degradation, cells were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

This blot was developed using a high-sensitivity ECL substrate, allowing for the detection of proteins in the mid-femtogram range.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-RPA32/RPA2 (phospho S33) antibody [EPR29612-551] (ab325750) at 1/1000 dilution

Lane 1:

Untreated NIH/3T3 (mouse embryonic fibroblast) fresh whole cell lysate at 50 µg

Lane 2:

NIH/3T3 treated with 1 μM camptothecin for 3 h fresh whole cell lysate at 50 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: 32 kDa,36 kDa,30 kDa

true

Exposure time: 70s

• Dot

Lab

Dot Blot - Anti-RPA32/RPA2 (phospho S33) antibody [EPR29612-551] (AB325750)

Dot blot analysis of RPA32/RPA2 (phospho S33) using ab325750 at 1 : 1000 (0.515 μg/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1 : 100,000 dilution.

Lane 1 : Human RPA32/RPA2 (phospho S33) peptide a

Lane 2 : Human RPA32/RPA2 (phospho S33) peptide b

Lane 3 : Human RPA32/RPA2 (non-phospho) peptide

Lane 4 : Human RPA32/RPA2 (phospho S39) peptide

Exposure time : 180 seconds.

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

All lanes:

Dot Blot - Anti-RPA32/RPA2 (phospho S33) antibody [EPR29612-551] (ab325750) at 1/1000 dilution

Lane 1:

Human RPA32/RPA2 (phospho S33) peptide a

Lane 2:

Human RPA32/RPA2 (phospho S33) peptide b

Lane 3:

Human RPA32/RPA2 (non-phospho) peptide

Lane 4:

Human RPA32/RPA2 (phospho S39) 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