- PA1-16565 - Invitrogen Antibodies TP53BP1 antibody

Submitted references Effect of Ionizing Radiation from Computed Tomography on Differentiation of Human Embryonic Stem Cells into Neural Precursors.

Hanu C, Loeliger BW, Panyutin IV, Maass-Moreno R, Wakim P, Pritchard WF, Neumann RD, Panyutin IG
International journal of molecular sciences 2019 Aug 10;20(16)

DNA damage response (DDR) pathway engagement in cisplatin radiosensitization of non-small cell lung cancer.

Sears CR, Cooney SA, Chin-Sinex H, Mendonca MS, Turchi JJ
DNA repair 2016 Apr;40:35-46

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Detection of Human (deleted mouse) 53BP1 by Western Blot Sample: Whole cell lysate (20 µg/lane) from U2OS or 293T cells resolved on a 3 to 8% tris-acetate gel. (Product # PA1-16565) used at 0.5 µg/mL.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of 53BP1 in total protein from HeLa, A431, Neuro2A, and PC12. Samples were incubated in 53BP1 polyclonal antibody (Product # PA1-16565) using a dilution of 1.0 µg/mL followed by an anti-rabbit HRP secondary antibody. Separated on a 12% gel by SDS-PAGE, transferred to PVDF membrane and blocked in 5% non-fat milk in TBST. Detection: chemiluminescence. The observed molecular weight for these samples are ~250 kDa and the theoretical molecular weight is 214 kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of 53BP1 in whole cell lysate from U2OS or 293T cells. Samples were incubated in 53BP1 polyclonal antibody (Product # PA1-16565). Bands indicate an observed molecular weight of ~220 kDa and the theoretical molecular weight is 214 kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockout validation by Western blot analysis of 53BP1 in immersion fixed HeLa cells (left) and 53BP1 knockout Hela cells (right). Samples were incubated in 53BP1 polyclonal antibody (Product # PA1-16565) using a dilution of 0.3 µg/mL for 3 hours at room temperature followed by a NorthernLights™ 557-conjugated Anti-Rabbit IgG secondary antibody. 53BP1 was detected in the fixed HeLa cells but was not detected in 53BP1 knockout Hela cells. Cells were counterstained with DAPI (blue). Specific staining was localized to nuclei.

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 # s14315, s14314). 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 # PA1-16565, 1:10000 ) 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 cell line models as observed here.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-53BP1 Polyclonal Antibody (Product # PA1-16565) and a ~270kDa 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), MDA-MB-231 (Lane 3), MCF7 (Lane 4), U-2 OS (Lane 5), K-562 (Lane 6), U-87 MG (Lane 7) 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 (1:10000 dilution) 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).A streak like pattern was observed in the positive cell lines which is expected of 53BP1 target.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunocytochemistry analysis of 53BP1 in proliferating MEFs. Samples were incubated in 53BP1 polyclonal antibody (Product # PA1-16565). Upper Panel: 53BP1 foci in proliferating MEFs. Lower Panel: 53BP1 foci in proliferating MEFs exposed to 10 Gy of IR.

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 # PA1-16565) at 1:1000 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 analysis of 53BP1 in formalin-fixed paraffin-embedded sections of human ovarian carcinoma (left) and mouse teratoma (right). Samples were incubated in 53BP1 polyclonal antibody (Product # PA1-16565) using a dilution of 1:1000. Detection: DAB.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemical analysis of 53BP1 in human colon cancer. Samples were incubated in 53BP1 polyclonal antibody (Product # PA1-16565) followed by DAB with hematoxylin counterstain.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometric detection of 53BP1. 1 million Jurkat cells were fixed, permeabilized, and stained with 1.5 µg/mL anti-53BP1 (Product # PA1-16565) in a 150 µl reaction. Isotype control (black), anti-53BP1 (red).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometry of 53BP1 in A431 cells. Samples were incubated in 53BP1 polyclonal antibody (Product # PA1-16565) using a dilution of 2.5 µg/mL for 30 minutes at room temperature. Antibody (blue) and a matched isotype control (orange). Cells were fixed with 4% PFA and then permeabilized with 0.1% saponin. Both antibodies were conjugated to Alexa Fluor 647.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

PA1-16565

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