MA5-12571

antibody from Invitrogen Antibodies

Targeting: TP53 LFS1, p53

Provider product page for MA5-12571

Western blot

Antibody data

Antibody Data
Antigen structure
References [68]
Comments [0]
Validations
Western blot [4]
Immunocytochemistry [3]
Immunoprecipitation [2]
Immunohistochemistry [1]
Other assay [9]

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Product number: MA5-12571 - Provider product page
Provider: Invitrogen Antibodies
Product name: p53 Monoclonal Antibody (DO-1)
Antibody type: Monoclonal
Antigen: Recombinant full-length protein
Description: MA5-12571 targets p53 in immunohistochemistry (paraffin) and Western blot applications and shows reactivity with Human samples.
Antibody clone number: DO-1
Concentration: 0.2 mg/mL

TP53 protein structure - MA5-12571 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of p53 was performed by loading 20 µg HeLa nuclear cell lysate per lane (untreated or treated with 20uM Camptothecin for 20 hours) and 7 µL of PageRuler Plus Prestained Protein Ladder (Product # 26619) per well onto a 4-20% Tris-Glycine polyacrylamide gel (Product # WT4202BX10). Proteins were transferred to a nitrocellulose membrane using the G2 Blotter (Product # 62288), and blocked with 5% Milk in TBST for 1 hour at room temperature. p53 was detected at ~53 kDa using a p53 mouse monoclonal antibody (Product # MA5-12571) at a concentration of 1 µg/mL in blocking buffer overnight at 4°C on a rocking platform, followed by a Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP conjugate (Product # 31430) at a dilution of 1:20000 for at least 30 minutes at room temperature. HDAC1 was detected using a HDAC1 rabbit polyclonal antibody (Product # PA1-860) at a concentration of 1 µg/mL. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34078).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of p53 was performed by loading 20 µg of indicated whole cell lysates per lane and 7 µL of PageRuler Plus Prestained Protein Ladder (Product # 26619) per well onto a 4-20% Tris-Glycine polyacrylamide gel (Product # WT4202BX10). Proteins were transferred to a nitrocellulose membrane using the G2 Blotter (Product # 62288), and blocked with 5% Milk in TBST for 1 hour at room temperature. p53 was detected at ~53 kDa using a p53 mouse monoclonal antibody (Product # MA5-12571) at a concentration of 1 µg/mL in blocking buffer overnight at 4°C on a rocking platform, followed by a Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP conjugate (Product # 31430) at a dilution of 1:20000 for at least 30 minutes at room temperature. GAPDH was detected using a GAPDH rabbit polyclonal antibody, HRP conjugate (Product # PA1-987-HRP) at a dilution of 1:1000 overnight at 4C on a rocking platform.Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34078).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-p53 Monoclonal Antibody (DO-1) (Product # MA5-12571) and a ~53 kDa band corresponding to TP53 was observed across cell lines tested . Whole cell extracts (30 µg lysate) of A-431 (Lane 1), T-47D (Lane 2), SK-O-V3 (Lane 3), HL-60 (Lane 4), U-937 (Lane 5) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177,1:20000) using the iBright™ FL1500 Imaging System (Product # A44115). Chemiluminescent detection was performed using SuperSignal™ West Atto Ultimate Sensitivity Substrate (Product # A38556).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockout of p53 was achieved by CRISPR-Cas9 genome editing (as confirmed by next generation sequencing) using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR718517_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of p53 was performed by loading 30 µg of HeLa Wild Type (Lane 1), Treated HeLa Wild type (Lane 2), HeLa Cas9 (Lane 3), Treated HeLa Cas9 (Lane 4), HeLa Cas9 cells transduced with p53 Lentiviral sgRNA (Lane 5), Treated HeLa Cas9 cells transduced with p53 Lentiviral sgRNA (Lane 6), HeLa p53 KO (Lane 7) and Treated HeLa p53 KO (Lane 8) whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Anti-p53 Monoclonal Antibody (DO-1) (Product # MA5-12571, 1:1,000 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4,000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to p53. * Treatment done with 5 µM Camptothecin for 20 hrs.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of TP53 was performed using 70% confluent log phase A-431 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with p53 Monoclonal Antibody (DO-1) (Product # MA5-12571) at 1:100 in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32766), (1:2000), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Green) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing nuclear localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 40X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of TP53 was performed using 70% confluent log phase T-47D cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with p53 Monoclonal Antibody (DO-1) (Product # MA5-12571) at 1:100 in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32766), (1:2000), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Green) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing nuclear localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 40X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of p53 (green) in untreated and 5uM 20 hours Camptothecin treated HeLa cells. The cells were fixed with 4% paraformaldehyde for 15 minutes at -20c, permeabilized with 0.1% Triton X-100 for 15 minutes, and blocked with 3% BSA for 30 minutes at room temperature. Cells were stained with a p53 mouse monoclonal antibody (Product # MA5-12571) at a concentration of 5 µg/mL in blocking buffer for 1 hour at room temperature, and then incubated with a Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor Plus 488 conjugate (Product # A32731) at a dilution of 1:500 for at least 30 minutes at a room temperature in the dark (green). F-actin (red) was stained by Dylight 554 Phalloidin (Product #21834) and nuclei (blue) were stained with Hoechst 33342 (Product # 62249). Images were taken on a Thermo Scientific ToxInsight Instrument at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 p53 accumulation and activation, caused by HUWE1 p.G4310R, perturb the cell cycle and impair proliferation in JMS patient-derived cells (A) Immunoblot analysis of the HUWE1, p53, and p21 protein levels in healthy control LCs and LCs from two JMS patients (JMS1 and JMS2). p53 protein levels relative to tubulin, serving as loading control, are indicated. (B) In vitro ubiquitination of purified recombinant p53 with increasing amounts of wild-type (WT) and p.G4310R HECT proteins. (C) In silico analysis of difference in the folding free energy change (DeltaDeltaG) of WT and G4310R HUWE1 using the indicated prediction tools. (D) Cell-cycle distribution determined by flow cytometry of healthy control, JMS1, and JMS2 LCs. (E) Fraction of annexin V-positive apoptotic cells measured by flow cytometry. (F) Proliferation rate of healthy control, JMS1, and JMS2 LCs. All error bars indicate mean +- SEM (n >= 3, biological replicates). Statistical significance was determined by two-way ANOVA with Tukey post-test (D) and Bonferroni post-test (F); one-way ANOVA with Bonferroni post-test; *p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5 AHNAK-regulated 53BP1 phase separation culminates in optimal p53 response (A) Schematic illustration of blue light-induced optoDroplet formation by 53BP1. (B) Representative image of Cry2-mCherry-53BP1-MFFR-BRCT-W1495A optoDroplet formation in WT and AHNAK -/- U2OS cells. After optoDroplet induction cells, were fixed and stained for p53 (fluorescein green) and DAPI (blue). (C) Quantification of sum 53BP1 optoDroplet fluorescence intensity in individual cells of the specified genotype. Solid line denotes mean. A.U., arbitrary units (WT, n = 222; DeltaAHNAK-1, n = 1,083; and DeltaAHNAK-2, n = 412). (D) Representative image of Cry2-mCherry optoDroplet formation in WT and AHNAK -/- U2OS cells transfected with CRY2 harboring or not the OD domain. Nuclei are depicted in blue (DAPI). (E) Quantification of sum 53BP1 optoDroplet fluorescence intensity in individual cells of the specified genotype from (D). Solid line denotes median. A.U., arbitrary units (n >= 2,650). (F) Schematic illustration of the LacO-LacI tethering system. (G) Co-localization of control GFP-LacI or GFP-53BP1-LacI (fluorescein green) with p53 (red) on the LacO array in U2OS19 cells after depletion with indicated siRNAs (n = 2 independent experiments). The white numbers represent the percentage of cells that exert colocalization of the indicated protein with the lacO array. (H) Fluorescence recovery after photobleaching (FRAP) analysis on the 53BP1-NBs. Graphical representation for normalized fluorescence

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6. DNA-RNA hybrid-dependent DSB foci formation and onset of DSB signalling. ( A ) Confocal imaging of MDC1 and GFP-RNaseH1 following transient transfection of pEGFP-M27 or non-transfected (mock) cells. n , number of cells with shown phenotype in %. ( B ) ChIP analysis of HA-53BP1 occupancy at DS1 using HA antibody (3F10) and site-specific primers. Asterisk, P -value < 0.05, two-tailed t -test. Error bar: mean +- SEM, n = 3. ( C ) Immunoblots detecting phospho-ATM, total ATM, gammaH2A.X, phospho-Chk1 and p53 levels following fluorescence-activated cell sorting (FACS) in absence of 4OHT (left) and after pulse-chase 4OHT incubation kinetics (right). Ponceau S, control.