Anti-p53 antibody [PAb 240]

• WB

Supplier Data

Western blot - Anti-p53 antibody [PAb 240] (AB26)

Lanes 1-6 : Merged (red and green) signal.

ab26 was shown to specifically react with p53 in wild type HCT116 cells treated with irinotecan. No band was observed in p53 knockout HCT116 cells. Wild-type and p53 knockout samples, positive and negative controls were subjected to SDS-PAGE. ab26 and ab181602 (loading control to GAPDH) were diluted 5 μg/mL and 1/10,000 respectively and incubated overnight at 4°C. Blots were developed with goat anti-rabbit IgG (H + L) and goat anti-mouse IgG (H + L) secondary antibodies at 1/10,000 dilution for 1 h at room temperature before imaging.

Wild-type and p53 knockout HCT116 cell lysates were kindly provided by a collaborator.

All lanes:

Western blot - Anti-p53 antibody [PAb 240] (ab26) at 5 µg/mL

Lane 1:

Wild-type HCT116 cell lysate at 30 µg

Lane 2:

Wild-type HCT116 + irinotecan (10 µM, 24 hours) cell lysate at 30 µg

Lane 3:

p53 knockout HCT116 cell lysate at 30 µg

Lane 4:

p53 knockout HCT116 + irinotecan (10 µM, 24 hours) cell lysate at 30 µg

Lane 5:

A431 cell lysate (positive control) at 20 µg

Lane 6:

Saos-2 cell lysate (negative control) at 20 µg

Predicted band size: 43 kDa

Observed band size: 53 kDa

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• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-p53 antibody [PAb 240] (AB26)

ab26 stained in A431 cells. Cells were fixed with 4% paraformaldehyde (10min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% triton for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with ab26 at 1μg/ml and ab6046 (Rabbit polyclonal to beta tubulin) at 1ug/ml overnight at +4°C. The secondary antibodies were ab150177 (colored green) used at 1 ug/ml and ab150087 (pseudo-colored red) used at 2ug/ml for 1hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43μM for 1hour at room temperature.

• IP

AbReview41852****

Immunoprecipitation - Anti-p53 antibody [PAb 240] (AB26)

p53 was immunoprecipitated from 7x10⁶ HCT116 (human colon carcinoma cell line) cells with ab26 at 1/150 dilution. Western blot was performed from the immunoprecipitate using anti-p53 antibody. Donkey Anti-Mouse IgG H&L (Alexa Fluor® 750) preadsorbed (ab175739) was used as secondary antibody at 1/5000 dilution.

Lane 1 : HCT116 whole cell lysate 10 μg (Input).

Lane 2 : ab207799 IP in etoposide treated HCT116 whole cell lysate.

Lane 3 : ab207799 IP in etoposide treated HCT116 p53-/- whole cell lysate (negative control).

All lanes:

Anti p53 antibody

Secondary

All lanes:

Immunoprecipitation - Donkey Anti-Mouse IgG H&L (Alexa Fluor® 750) preadsorbed (<a href='/en-us/products/secondary-antibodies/donkey-mouse-igg-h-l-alexa-fluor-750-preadsorbed-ab175739'>ab175739</a>) at 1/5000 dilution

Predicted band size: 43 kDa

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• WB

Project

Western blot - Anti-p53 antibody [PAb 240] (AB26)

All lanes:

Western blot - Anti-p53 antibody [PAb 240] (ab26) at 5 µg/mL

Lane 1:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 20 µg

Lane 2:

Hela Whole Cell Lysate - Bleomycin Treated (40U/ml) at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Mouse IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-hrp-preadsorbed-ab97040'>ab97040</a>) at 1/5000 dilution

Predicted band size: 43 kDa

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Exposure time: 4min

• WB

Lab

Western blot - Anti-p53 antibody [PAb 240] (AB26)

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab26 overnight at 4°C. Antibody binding was detected using an anti-mouse HRP secondary antibody (ab97040), and visualised using ECL development solution ab133406.

All lanes:

Western blot - Anti-p53 antibody [PAb 240] (ab26) at 1 µg/mL

All lanes:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Mouse IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-hrp-preadsorbed-ab97040'>ab97040</a>) at 1/50000 dilution

Predicted band size: 43 kDa

Observed band size: 53 kDa

true

Exposure time: 8min

• WB

AbReview11453****

Western blot - Anti-p53 antibody [PAb 240] (AB26)

All lanes:

Western blot - Anti-p53 antibody [PAb 240] (ab26) at 1/2000 dilution

Lane 1:

Human breast cancer cell-line, MCF7 cells (p53 WT), whole cell lysate at 20 µg

Lane 2:

Human breast cancer cell-line, MDA231 cells (p53 Mutant), whole cell lysate at 20 µg

Secondary

All lanes:

HRP conjugated donkey anti-mouse antibody at 1/5000 dilution

Predicted band size: 43 kDa

Observed band size: 53 kDa,72 kDa

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Exposure time: 10s

This image is courtesy of an Abreview submitted by Dr Cherie Blenkiron

• WB

Lab

Western blot - Anti-p53 antibody [PAb 240] (AB26)

Lanes 1-2 : 1% BSA blocking buffer

Lanes 3-4 : 3% Milk blocking buffer

We recommend using 3% milk as the blocking agent for Western blot.

Lanes 1 - 2:

Western blot - Anti-p53 antibody [PAb 240] (ab26) at 1 µg/mL

Lanes 3 - 4:

Western blot - Anti-p53 antibody [PAb 240] (ab26) at 5 µg/mL

All lanes:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 20 µg

Secondary

All lanes:

Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/5000 dilution

Predicted band size: 43 kDa

true

Exposure time: 4min

• WB

Unknown

Western blot - Anti-p53 antibody [PAb 240] (AB26)

Primary : All Lanes : Anti-p53 antibody (ab26) at 5 μg/mL. Lane 1 : MW marker. Lane 2 : NIH/3T3 cells treated with vehicle for 24 hours. Lane 3 : NIH/3T3 cells treated with 1 μM doxorubicin for 24 hours Secondary : All Lanes : HRP-conjugated VeriBlot anti-Mouse IgG (ab131368) 1 : 1000. Lysates at 20 μg/lane. Performed under denaturing conditions. Developed using ECL technique. Blocking buffer : 5% milk in PBS.

All lanes:

Western blot - Anti-p53 antibody [PAb 240] (ab26)

Predicted band size: 43 kDa

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• WB

CiteAb

Western blot - Anti-p53 antibody [PAb 240] (AB26)

Western Blotting using Anti-p53 antibody [PAb 240], ab26. Publication image from Zhang, P. et al., 2020, J Biomed Sci, 32005234. Legend direct from paper.

SERPINB5 is indispensable for TRIM21-mediated GMPS–TP53 repression after radiation. a Co-immunoprecipitation following western blotting in NPC cells with SERPINB5–HA and TRIM21–MYC overexpression. b Immunofluorescence staining analysis of SERPINB5 and TRIM21 in NPC cells. c Western blot detection of SERPINB5 expression in normal NP69 cell line and in NPC cell lines. d Western blot detection of GMPS and TP53 in NPC cells with TRIM21 GOF or SERPINB5 LOF. e Co-immunoprecipitation using anti-HA antibody in NPC cells with SERPINB5–HA and GMPS–FLAG overexpression. f Immunofluorescence staining to detect the co-localization of GMPS and SERPINB5 in NPC cells with or without ionizing radiation. g GMPS expression in immune-purified protein by anti-HA antibody from NPC cells with or without TRIM21 LOF. h GMPS expression in immune-purified protein by anti-MYC antibody from NPC cells with or without SERPINB5 LOF. i GMPS expression in NPC cells with TRIM21 or SERPINB5 overexpression. j Immunofluorescence staining of overexpressed GMPS and ubiquitin in HONE1 cells with or without ionizing radiation. The localization of GMPS and was evaluated in condition of SERPINB5 or TRIM21 LOF. For the co-immunoprecipitation assay, MG132 was added into the cell medium before cell harvesting to avoid GMPS degradation. *P < 0.05, **P < 0.01, ***P < 0.001

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• WB

CiteAb

Western blot - Anti-p53 antibody [PAb 240] (AB26)

Western Blotting using Anti-p53 antibody [PAb 240], ab26. Publication image from Zhang, P. et al., 2020, J Biomed Sci, 32005234. Legend direct from paper.

TRIM21 protects NPC cells from radiation-induced apoptosis by manipulating the GMPS–TP53 cascade. a Western blot detection of TP53 expression in HONE1 cells after radiation. b Co-immunoprecipitation using anti-FLAG antibody following western blot detection of USP7 and TP53 expression in GMPS–FLAG-overexpressing NPC cells with or without X-ray radiation. c GMPS expression in TRIM21-overexpressing NPC cells with or without X-ray radiation. d Co-immunoprecipitation using anti-MYC antibody following western blot detection of GMPS expression in TRIM21–MYC-overexpressing NPC cells with or without X-ray radiation. e Flow cytometry analysis of Annexin V and PI staining in HONE1 cells with TRIM21 GOF or LOF after X-ray radiation. Annexin+PI− cells, which were in the early apoptotic stage, were evaluated and quantified. f, g Quantification of the apoptotic HONE1 f and 5-8F g cells. h, i Clonogenic survival assay of HONE1 cells with TRIM21 GOF h or LOF i. j Absorbance intensity of TRIM21 GOF and LOF tumor cells and their counterpart controls in mice (n = 5 for each group). The tumors were evaluated 2 and 3 weeks, respectively, after implantation, and the mice received radiotherapy (2 Gy daily and a total of 12 Gy) after 2 weeks. k, l The absorbance intensity analysis of tumors in mice. For the co-immunoprecipitation assay, the cells were pre-treated with MG132 to avoid GMPS degradation before harvesting. *P < 0.05, **P < 0.01, ***P < 0.001. Mu, mutant; ns, not significant; IP, immunoprecipitation

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• WB

CiteAb

Western blot - Anti-p53 antibody [PAb 240] (AB26)

Western Blotting using Anti-p53 antibody [PAb 240], ab26. Publication image from Zhang, T. et al., 2022, Nat Commun, 36376321. Legend direct from paper.

Kmt5b prevents replication stress and genome instability in proliferating MuSC.a Proliferation rate of Ctrl (n = 3 mice) and Kmt5bsKO (n = 4 mice) MuSCs quantified during time lapse imaging. Confluency of Ctrl and Kmt5bsKO MuSCs at the last time point is on the right. b Immunofluorescence staining for EdU and γH2AX in Ctrl and Kmt5bsKO MuSCs after 4 days in culture (n = 4 mice). DNA was stained by DAPI. Scale bar : 10 µm. Median of fluorescence intensity (MFI, arbitrary units (arb. units)) of γH2AX/EdU+ and γH2AX/EdU− MuSCs are shown on the right. Data are shown as mean ± SEM; Two-way ANOVA. c Representative images of DNA fibers labeled with CIdU and IdU. Scale bar : 10 µm. Quantification of fork progression in Ctrl and Kmt5bsKO MuSCs is shown on the right (n = 3 mice). Data are shown as mean ± SEM; Two-way. d Percentage of stalled folks in Ctrl and Kmt5bsKO MuSCs in all fibers (n = 3 mice). e Images of DAPI-stained metaphase chromosome spreadings prepared from Ctrl and Kmt5bsKO MuSCs (n = 3 mice). Scale bar : 10 µm. Quantification of aneuploid MuSCs is on the right. f Immunofluorescence staining forα-tubulin/DAPI showing Kmt5bsKO MuSCs with micronuclei (indicated by arrowheads). Scale bar : 10 µm. Percentage of Ctrl (n = 3 mice) or Kmt5bsKO (n = 4 mice) MuSCs with micronuclei is on the right. g Western blot analysis of pP53 and γH2AX in Ctrl and Kmt5bsKO MuSCs after 4 days in culture. Pan-Actin was used as loading control. Band intensities were quantified, corresponding plots are shown on the right (n = 3 mice). h RT-qPCR analyses of p21 expression in Ctrl and Kmt5bsKO MuSCs (n = 3 mice). 36b4 was used as reference gene. i SA-β-GAL staining of Ctrl and Kmt5bsKO MuSCs after 7-days in proliferation medium (n = 3 mice). Scale bar : 20 µm. Quantification of SA-β-GAL positive cells is on the right. Data are shown as mean ± SEM. Unpaired two-sided Student’s t-tests were employed for all panels except for (b) and (c). Source data are provided in the Source Data file.

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