- PA5-54565 - Invitrogen Antibodies TP53BP1 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of 53BP1 in Lane 1: Marker (kDa) 230, 130, 95, 72, 56, 36, 28, 17, 11; Lane 2: Human cell line RT-4; Lane 3: Human cell line U-251MG sp. Samples were probed using a 53BP1 Polyclonal Antibody (Product # PA5-54565).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of 53BP1 in Lane 1: NIH-3T3 cell lysate (Mouse embryonic fibroblast cells); Lane 2: NBT-II cell lysate (Rat Wistar bladder tumour cells). Samples were probed using a 53BP1 Polyclonal Antibody (Product # PA5-54565).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of TP53-binding protein 1 was achieved by transfecting HeLa with TP53-binding protein 1 specific siRNAs (Silencer® select Product # Retrieving data. Wait a few seconds and try to cut or copy again.). Western Blot analysis (Fig. a) was performed using Nuclear enriched extracts from the TP53-binding protein 1 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with 53BP1 Polyclonal Antibody (Product # PA5-54565, 0.04 µg/mL ) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000). Densitometric analysis of this Western Blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to TP53-binding protein 1.53BP1 appears like a streak in positive models as observed.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot was performed using Anti-53BP1 Polyclonal Antibody (Product # PA5-54565) and a ~270 kDa band corresponding to TP53-binding protein 1 was observed across cell lines tested . Nuclear enriched extracts (30 µg lysate) of HeLa (Lane 1), A549 (Lane 2), MCF7 (Lane 3), U-2 OS (Lane 4), K-562 (Lane 5), U-87 MG (Lane 6) were electrophoresed using NuPAGE™ 3-8% Tris-Acetate Protein Gel (Product # EA0378BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # LC2001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (0.05 µg/mL) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).53BP1 picks up in a streak pattern as observed in the positive cell lines here.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent staining of 53BP1 in human cell line A-431 shows positivity in nucleus. Samples were probed using a 53BP1 Polyclonal Antibody (Product # PA5-54565).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of TP53-binding protein 1 was performed using 70% confluent log phase HeLa 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 53BP1 Polyclonal Antibody (Product # PA5-54565) at 0.5 µg/mL in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), (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: Red) 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 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemical staining of 53BP1 in human cerebellum tissue shows strong nuclear positivity. Samples were probed using a 53BP1 Polyclonal Antibody (Product # PA5-54565).

PA5-54565