Anti-p53 antibody [G59-12] - BSA and Azide free

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-p53 antibody [G59-12] - BSA and Azide free (AB308610)

This data was developed using ab308609, the same antibody clone in a different buffer formulation.

ab308609 was shown to react with TP53 in wild-type MCF7 cells in immunocytochemistry with loss of signal observed in TP53 knockout cell line ab286440. Wild-type and knockout cells were mixed and pelleted at a 1 : 1 ratio on coverslips. The cells were fixed with 4% paraformaldehyde (15 min) then permeabilized with 0.1% Triton X-100 (10min) and then blocked with 1x PBS, 0.01% Triton X-100, 5% BSA, 5% NGS. The cells were then incubated with ab308609 at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat anti-rabbit secondary antibody to (Alexa Fluor® 555) at 0.5 μg/ml. Acquisition of the green (wild-type), red (antibody staining) and far-red (knockout) channels was performed. Representative grayscale images of the red channel are shown. Wild-type and knockout cells are outlined with yellow and magenta dashed line, respectively. Schematic representation of the mosaic strategy used is shown on the bottom-right panel. Image was acquired with a Zeiss(LSM-880).

This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p53 antibody [G59-12] - BSA and Azide free (AB308610)

This data was produced using ab308609, the same antibody clone but in a different buffer. Immunohistochemical analysis of paraffin-embedded human cardiac muscle tissue labeling p53 with ab308609 at 1/100 (10.28 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Negative control : No staining on human cardiac muscle. The section was incubated with ab308609 for 30 mins at room temperature, followed by anti-mouse IgG1 antibody (ab125913) for 8 mins during the LeicaDS9800 kit staining procedure. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p53 antibody [G59-12] - BSA and Azide free (AB308610)

This data was produced using ab308609, the same antibody clone but in a different buffer. Immunohistochemical analysis of paraffin-embedded cells labeling p53 with ab308609 at 1/100 (10.28 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Mainly nuclear staining on (A) wild-type HAP1 cell pellet, no staining on (B) p53 knockout HAP1 cell pellet. The section was incubated with ab308609 for 30 mins at room temperature, followed by anti-mouse IgG1 antibody (ab125913) for 8 mins during the LeicaDS9800 kit staining procedure. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p53 antibody [G59-12] - BSA and Azide free (AB308610)

This data was produced using ab308609, the same antibody clone but in a different buffer. Immunohistochemical analysis of paraffin-embedded human endometrial cancer tissue labeling p53 with ab308609 at 1/100 (10.28 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Mainly nuclear staining on human endometrial cancer. The section was incubated with ab308609 for 30 mins at room temperature, followed by anti-mouse IgG1 antibody (ab125913) for 8 mins during the LeicaDS9800 kit staining procedure. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-p53 antibody [G59-12] - BSA and Azide free (AB308610)

This data was produced using ab308609, the same antibody clone but in a different buffer. Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized p53 KO HAP1 (human p53 knockout chronic myelogenous leukemia near-haploid cell, Left) / Parental HAP1 (Right) cells labelling p53 with ab308609 at 1/1000 dilution (0.1 ug) (Red) compared with a Mouse monoclonal IgG (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Mouse IgG (Alexa Fluor® 647, ab150119) at 1/5000 dilution was used as the secondary antibody.

• IP

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Immunoprecipitation - Anti-p53 antibody [G59-12] - BSA and Azide free (AB308610)

This data was developed using ab308609, the same antibody clone in a different buffer formulation.

Immunoprecipitation of TP53 in HAP1 cells. Lysates were prepared and immunoprecipitation was performed using 2 μg of ab308609 pre-coupled to Protein A beads. Samples were then washed and processed for western blot.

This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Immunoprecipitation - Anti-p53 antibody [G59-12] (<a href='/ko/products/primary-antibodies/p53-antibody-g59-12-ab308609'>ab308609</a>) at 2 µg

All lanes:

HAP1 cells

false

• IP

Supplier Data

Immunoprecipitation - Anti-p53 antibody [G59-12] - BSA and Azide free (AB308610)

This data was produced using ab308609, the same antibody clone but in a different buffer. p53 was immunoprecipitated from 0.35 mg HEK-293 (human embryonic kidney epithelial cell) whole cell lysate with ab308609 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab308609 at 1/1000 dilution. mouse IgG for IP (HRP) (ab131368) was used at 1/5000 dilution. Lane 1 : HEK-293 whole cell lysate Lane 2 : ab308609 IP in HEK-293 whole cell lysate Lane 3 : Mouse IgG1 monoclonal isotype control (ab18443) instead of ab308609 in HEK-293 whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 23 seconds. The binding pattern observed is consistent with what has been described in the literature (PMID : 31045216; PMID : 16131611).

All lanes:

Immunoprecipitation - Anti-p53 antibody [G59-12] (<a href='/ko/products/primary-antibodies/p53-antibody-g59-12-ab308609'>ab308609</a>) at 1/1000 dilution

All lanes:

HEK-293 whole cell lysate at 10 µg

Secondary

All lanes:

Immunoprecipitation - Anti-mouse IgG for IP (HRP) (<a href='/ko/products/secondary-antibodies/mouse-igg-for-ip-hrp-ab131368'>ab131368</a>) at 1/5000 dilution

false

• WB

Supplier Data

Western blot - Anti-p53 antibody [G59-12] - BSA and Azide free (AB308610)

This data was produced using ab308609, the same antibody clone but in a different buffer. Blocking and diluting buffer and concentration : 5% NFDM/TBST. Negative control : Saos-2 (PMID : 17202229; PMID : 9786925). In Western blot, anti- Vinculin antibody (ab129002) loading control staining at 1/10000 dilution. Exposure time : 180 seconds.

All lanes:

Western blot - Anti-p53 antibody [G59-12] (<a href='/ko/products/primary-antibodies/p53-antibody-g59-12-ab308609'>ab308609</a>) at 1/1000 dilution

Lane 1:

HEK-293 (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 2:

Saos-2 (human osteosarcoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/10000 dilution

Observed band size: 53 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-p53 antibody [G59-12] - BSA and Azide free (AB308610)

This data was developed using ab308609, the same antibody clone in a different buffer formulation.

ab308609 was shown to react with TP53 in wild-type HAP1 cells in Western blot with loss of signal observed in a TP53 knockout cell line. Wild-type HAP1 and TP53 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab308609 overnight at 4 °C at a 1/1000 dilution. Blots were incubated with secondary antibodies at 0.2 µg/mL before imaging.

This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Western blot - Anti-p53 antibody [G59-12] (<a href='/ko/products/primary-antibodies/p53-antibody-g59-12-ab308609'>ab308609</a>) at 1/1000 dilution

Lane 1:

Wild-type HAP1 lysate at 20 µg

Lane 2:

TP53 knock-out HAP1 lysate at 20 µg

false

• WB

Supplier Data

Western blot - Anti-p53 antibody [G59-12] - BSA and Azide free (AB308610)

This data was produced using ab308609, the same antibody clone but in a different buffer. Blocking and diluting buffer and concentration : Intercept® (TBS) Blocking Buffer diluted with an equal volume of TBS. Lysates at 20 µg per lane. The samples were run on a Bis-Tris gel under reducing conditions. Western blot : Anti-p53 antibody (ab308609) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody (ab181602) loading control staining at 1/2000 dilution, shown in red. In Western blot, ab308609 was shown to bind specifically to p53. Target of interest was observed at 53 kDa in wild-type HAP1 cell lysates (lane 1) with no signal observed at this size in p53 knockout cell lysates (lane 2). To generate this image, samples were first run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane.  Membranes were blocked in a fluorescent western blot (TBS-based) blocking solution before incubation with primary antibodies overnight at 4 °C.  Blots were washed in TBS-T, incubated with secondary antibodies Goat Anti-Mouse IgG H&L (IRDye® 800CW) and Goat Anti-Rabbit IgG H&L (IRDye® 680RD) at 1/20000 dilution for 1 h at room temperature, washed again then imaged.

All lanes:

Western blot - Anti-p53 antibody [G59-12] (<a href='/ko/products/primary-antibodies/p53-antibody-g59-12-ab308609'>ab308609</a>) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

p53 knockout HAP1 whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (<a href='/ko/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-800cw-preadsorbed-ab216772'>ab216772</a>) at 1/20000 dilution

Observed band size: 53 kDa

false