Anti-p53 (acetyl K305 + K370 + K373) antibody [RM1200]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-p53 (acetyl K305 + K370 + K373) antibody [RM1200] (AB321819)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa (human cervical adenocarcinoma epithelial cell) cells labelling p53 (acetyl K305 + K370 + K373) with ab321819 at 1/2000 (0.261 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 Doxorubicin (0.5 µM) for 24 h and further increased signal treated with Doxorubicin (0.5 µM) and Trichostatin A (500 ng/mL) for 24 hr. The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-p53 (acetyl K305 + K370 + K373) antibody [RM1200] (AB321819)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Untreated HeLa (human cervical adenocarcinoma epithelial cell) (Black) / HeLa treated with 0.5µM Doxorubicin for 24h (Megenta) / HeLa treated with 0.5µM Doxorubicin and 500ng/mL Trichostatin A for 24h (Green) cells labelling p53 (acetyl K305 + K370 + K373) with ab321819 at 1/50 dilution (1ug) / Black, Megenta and Green compared with a isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-p53 (acetyl K305 + K370 + K373) antibody [RM1200] (AB321819)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling p53 (acetyl K305 + K370 + K373) with ab321819 at 1/2000 (0.261 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 NIH/3T3 cells (shown in green) treated with Doxorubicin (0.5 µM) for 24 h and further increased signal treated with Doxorubicin (0.5 µM) and Trichostatin A (500 ng/mL) for 24 hr. The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-p53 (acetyl K305 + K370 + K373) antibody [RM1200] (AB321819)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Untreated NIH/3T3 (mouse embryonic fibroblast) (Black) / NIH/3T3 treated with 0.5µM Doxorubicin for 24h (Megenta) / NIH/3T3 treated with 0.5µM Doxorubicin and 500ng/mL Trichostatin A for 24h (Green) cells labelling p53 (acetyl K305 + K370 + K373) with ab321819 at 1/50 dilution (1ug) / Black, Megenta and Green compared with a isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• IP

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Immunoprecipitation - Anti-p53 (acetyl K305 + K370 + K373) antibody [RM1200] (AB321819)

p53 (acetyl K305 + K370 + K373) was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast) whole cell lysate with ab321819 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab321819 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : NIH/3T3 (mouse embryonic fibroblast) whole cell lysate
Lane 2 : ab321819 IP in NIH/3T3 (mouse embryonic fibroblast) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab321819 in NIH/3T3 whole cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST

All lanes:

Immunoprecipitation - Anti-p53 (acetyl K305 + K370 + K373) antibody [RM1200] (ab321819) at 1/30 dilution

All lanes:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 24s

• WB

Supplier Data

Western blot - Anti-p53 (acetyl K305 + K370 + K373) antibody [RM1200] (AB321819)

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

Negative control : Saos-2 (PMID : 25490093)

The expression of acetyl p53 is upregulated in response to doxorubicin and TSA treatment (PMID : 15123817).

The identity of the lower MW band at approximately 15 kDa is unknown.

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

All lanes:

Western blot - Anti-p53 (acetyl K305 + K370 + K373) antibody [RM1200] (ab321819) at 1/1000 dilution

Lane 1:

Untreated HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

HeLa treated with 0.5µM Doxorubicin (<a href='/en-us/products/biochemicals/doxorubicin-hydrochloride-topoisomerase-ii-inhibitor-ab120629'>ab120629</a>) for 24 hours whole cell lysate at 20 µg

Lane 3:

HeLa treated with 0.5µM Doxorubicin and 500ng/ml TSA(Trichostatin A) for 24 hours whole cell lysate at 20 µg

Lane 4:

Saos-2 (human osteosarcoma epithelial cell) 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 1/100000 dilution

Observed band size: 53 kDa,36 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-p53 (acetyl K305 + K370 + K373) antibody [RM1200] (AB321819)

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

The expression of acetyl p53 is upregulated in response to doxorubicin and TSA treatment (PMID : 15123817).

The identity of the lower MW band at approximately 15 kDa is unknown.

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

All lanes:

Western blot - Anti-p53 (acetyl K305 + K370 + K373) antibody [RM1200] (ab321819) at 1/1000 dilution

Lane 1:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 2:

NIH/3T3 treated with 0.5µM Doxorubicin (<a href='/en-us/products/biochemicals/doxorubicin-hydrochloride-topoisomerase-ii-inhibitor-ab120629'>ab120629</a>) for 24 hours whole cell lysate at 20 µg

Lane 3:

NIH/3T3 treated with 0.5µM Doxorubicin and 500ng/ml TSA(Trichostatin A) for 24 hours 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 1/100000 dilution

Observed band size: 53 kDa,36 kDa

false

Exposure time: 180s

• Dot

Supplier Data

Dot Blot - Anti-p53 (acetyl K305 + K370 + K373) antibody [RM1200] (AB321819)

Dot blot analysis of p53 (acetyl K305 + K370 + K373) using ab321819 at 1 : 1000 (0.521 ug/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1 : 100,000 dilution.

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

All lanes:

Dot Blot - Anti-p53 (acetyl K305 + K370 + K373) antibody [RM1200] (ab321819) at 1/1000 dilution

Lane 1:

Human p53 (acetyl K305) peptide

Lanes 2, 4 and 6:

Human p53 non-acetyl peptide

Lane 3:

Human p53 (acetyl K370) peptide

Lane 5:

Human p53 (acetyl K373) 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