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Anti 53bp1

Manufactured by Santa Cruz Biotechnology
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Anti-53BP1 is a laboratory reagent used to detect the 53BP1 protein in biological samples. 53BP1 is a DNA damage response protein that plays a role in the repair of double-strand breaks in DNA. The Anti-53BP1 product is designed for use in techniques such as Western blotting, immunohistochemistry, and immunofluorescence to identify and quantify the presence of 53BP1 in cells or tissues.

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Lab products found in correlation

Anti γh2ax, by Merck Group (3 mentions) Anti p53, by Santa Cruz Biotechnology (3 mentions) Anti α tubulin, by Merck Group (3 mentions) Anti mer11, by Santa Cruz Biotechnology (2 mentions) Anti γ h2ax, by Santa Cruz Biotechnology (2 mentions) Anti atr, by Santa Cruz Biotechnology (2 mentions) Nocodazole, by Merck Group (1 mentions) Doxycycline, by Merck Group (1 mentions) Sir dna kit, by Spirochrome (1 mentions) Bromodeoxyuridine (brdu), by Merck Group (1 mentions) Anti cyclin e1, by Cell Signaling Technology (1 mentions) Anti prb, by Cell Signaling Technology (1 mentions) Hoechst 33342, by Merck Group (1 mentions) Pimonidazole, by Hypoxyprobe (1 mentions) Anti uqcrc2, by Abcam (1 mentions) Anti ndufa9, by Abcam (1 mentions) Anti cytochrome c, by Santa Cruz Biotechnology (1 mentions) Anti tom20, by Santa Cruz Biotechnology (1 mentions) Anti ant1, by Abcam (1 mentions) Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)

Close spelling variants of this product

Rabbit anti-53BP1(#sc-22760) Anti-53BP1 rabbit polyclonal antibody Anti-53BP1 (sc-22760; Anti-53BP1 (H-300) (sc-22760, Anti-53BP1 antibody Primary anti-53BP1 antibody

12 protocols using anti 53bp1

1

Immunofluorescence Analysis of DNA Damage Markers

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Antibodies used in the study: anti-53BP1 (dilution: 1:500, sc-629, Santa Cruz Biotechnology), anti-γH2AX (dilution: 1:1000, 05-636, Upstate Biotechnology, Millipore), secondary antibodies (dilution: 1:600, anti-mouse Alexa 568, A11004, anti-rabbit Alexa 488, A11008, Molecular Probes), anti-pimonidazole (mouse monoclonal 4.3.11.3, Natural Pharmacia International, Belmont, MA, USA, dilution 1:100), anti-BrdU (mouse monoclonal, Clone Bu20a, Dako Deutschland GmbH, Hamburg, Germany, dilution: 1:50), anti-pRB (Ser807/811, 1/1000) (Cell signaling, #9308), anti-Cyclin E1 (Cell signaling, 20808, 1/1000), anti-HIF1a (Cayman Chemicals 10006421, 1/1000), anti-alpha-tubulin (Sigma Aldrich T5168, 1/1000). Reagents used in the study: Doxycycline (D9891, Sigma-Aldrich), nocodazole (M1404, Sigma-Aldrich), Hoechst 33342 (B2261, Sigma-Aldrich), BrdU (Sigma 850187), pimonidazole (Hypoxyprobe Inc, hpi, Middlesex, Burlington, USA), SirDNA kit (SPIROCHROME), AEC kit (Signa Aldrich AEC 101), Dako Faramount aqueous mounting medium (S3025).
Menegakis A., Klompmaker R., Vennin C., Arbusà A., Damen M., van den Broek B., Zips D., van Rheenen J., Krenning L, & Medema R.H. (2021). Resistance of Hypoxic Cells to Ionizing Radiation Is Mediated in Part via Hypoxia-Induced Quiescence. Cells, 10(3), 610.
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2

Protein Immunoblotting Assay Protocol

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All chemicals were purchased from Sigma (St. Louis, MO, USA). For immunoblotting, the following antibodies were used: anti-ANT2, anti-cleaved caspase-3 (Cell Signaling Technology, Danvers, MA, USA), anti-Tom20, anti-cytochrome C, anti-53BP1 (Santa Cruz Biotechnology, Dallas, TX, USA), anti-ATP5B (Sigma, St. Louis, MO, USA), anti-γH2AX (Millipore, Billerica, MA, USA), anti-NDUFA9, anti-SDHA, anti-Cox5a, anti-UQCRC2, anti-ANT1 and anti-VDAC (Abcam, Cambridge, UK). All antibodies were diluted 1:1000 in 2.5% non-fat milk. Horseradish peroxidase (HRP) conjugated β-actin (ThermoFisher, Waltham, MA, USA) was used as a loading control. IgG-HRP anti-rabbit (170-6515) and anti-mouse (170-6516) secondary antibodies produced in goat were purchased from BioRad Laboratories (Hercules, CA, USA). Secondary antibodies were diluted 1:10,000 in 2.5% non-fat milk.
Hubackova S., Davidova E., Rohlenova K., Stursa J., Werner L., Andera L., Dong L., Terp M.G., Hodny Z., Ditzel H.J., Rohlena J, & Neuzil J. (2018). Selective elimination of senescent cells by mitochondrial targeting is regulated by ANT2. Cell Death and Differentiation, 26(2), 276-290.
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3

Immunofluorescence Assay for NF-κB and DNA Damage

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MEF cells were seeded to ~8.57x104 the day before using a hemocytometer. MEFs were incubated with NSC697923 for 30 minutes prior to either LPS stimulation or ionizing radiation treatment. Cells were fixed using 4 % paraformaldehyde and stained with either an anti-p65 antibody (Santa Cruz, sc-372), or anti-53BP1 (Santa Cruz, sc-22760) and anti-γH2AX (Millipore, 05–636) antibodies. Invitrogen (37–1100) anti-Ubc13 antibody was used for Western blotting. MetaMorph was used to acquire single-plane images. Images were independently scaled in Photoshop CS3 (Adobe) for Windows, to best represent the subcellular distribution of the fluorescent stain. Further technical details are described in Supporting Information.
Hodge C.D., Edwards R.A., Markin C.J., McDonald D., Pulvino M., Huen M.S., Zhao J., Spyracopoulos L., Hendzel M.J, & Glover J.N. (2015). Covalent Inhibition of Ubc13 Affects Ubiquitin Signaling and Reveals Active Site Elements Important for Targeting. ACS chemical biology, 10(7), 1718-1728.
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4

Immunofluorescent Visualization of DNA Damage Markers

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Cells grown on coverslips were fixed in 1% paraformaldehyde in methanol on ice for 10 minutes. Fixed cells were immunofluorescently labeled with the following primary and secondary antibodies:anti-53BP1 (rabbit polyclonal, 1:200) (Santa Cruz Biotechnology: sc-22760), anti-γH2AX-S139 (mouse monoclonal, 1:200) (EMD Millipore: 05-636), goat anti-mouse IgG (H+L) Alexa Fluor 594, and goat anti-rabbitIgG (H+L) Alexa Fluor 488. The nuclei were counterstained with DAPI. Cells were imaged on a Zeiss AxioImager. Z2 equipped with Hamamatsu CCD camera, and images were captured using Zen2012 software. Image analyses were performed using ImageJ (https://imagej.nih.gov/).
Tracy K.M., Tye C.E., Ghule P.N., Malaby H.L., Stumpff J., Stein J.L., Stein G.S, & Lian J.B. (2018). Mitotically-associated lncRNA (MANCR) Affects Genomic Stability and Cell Division in Aggressive Breast Cancer. Molecular cancer research : MCR, 16(4), 587-598.
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5

Immunofluorescent Analysis of DNA Damage

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HSPCs were fixed with 4% paraformaldehyde and treated with anti-phospho-H2AX (Millipore, MA, USA) or anti-53BP1 (Santa Cruz Biotechnology, CA, USA), which was diluted in blocking buffer (1% BSA/0.1% Triton X-100). Thereafter, either anti-mouse-PE or anti-rabbit-FITC (BD Bioscience) was added as a secondary antibody (1:500). The nuclei were stained with DAPI from Sigma (St. Louis, MO, USA).
Hwang Y.J., Shin D.Y., Kim M.J., Jang H., Kim S., Yang H., Jang W.I., Park S., Shim S, & Lee S.B. (2023). StemRegenin 1 Mitigates Radiation-Mediated Hematopoietic Injury by Modulating Radioresponse of Hematopoietic Stem/Progenitor Cells. Biomedicines, 11(3), 824.
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6

Immunofluorescence Imaging of DNA Damage Markers

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Immunofluorescence was performed as previously reported [51 (link)]. Cells were fixed in 2% formaldehyde and permeabilized in 0.25% Triton X100 in PBS for 5 min at room temperature. For immunolabeling, cells were incubated with primary antibody, washed in PBS and incubated with fluorophore-conjugated secondary antibodies. The following monoclonal primary antibodies were used: anti-TRF1, anti-ATR, anti-γ-H2AX, anti-53BP1, anti-Mer11 and anti-hTERT, (Santa Cruz Biotechnology, Inc.). Rhodamine- or DyLightTM488-conjugated goat anti-rabbit and fluorescein- or DyLightTM594-conjugated goat anti-mouse (ZSGB-BIO) served as secondary antibodies. Fluorescence signals were captured using an Olympus Fluoview FV1000 confocal microscope and analyzed using the FV10-ASW 1.6 Viewer program (Olympus, Japan).
Cheng Y., Li Y., Ma C., Song Y., Xu H., Yu H., Xu S., Mu Q., Li H., Chen Y, & Zhao G. (2016). Arsenic trioxide inhibits glioma cell growth through induction of telomerase displacement and telomere dysfunction. Oncotarget, 7(11), 12682-12692.
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7

Immunofluorescence Assay Protocol

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Cells were fixed for 15 min in PBS-buffered 4% paraformaldehyde, followed by permeablization for 5 min in Triton-X buffer68 (link). The following antibodies were used at a concentration of 1–2 μg ml−1 for immunofluorescence assays: anti-CENP-B (sc22788; Santa Cruz Biotechnology), anti-BubR1 (612503), anti-α-tubulin (T9026; Sigma-Aldrich), anti-p53 (sc126; Santa Cruz Biotechnology), anti-53BP1 (sc22760; Santa Cruz Biotechnology), anti-p62 (sc28359; Santa Cruz Biotechnology) and anti-LC3B (3868; Cell Signaling Technology). Images were captured with a Plan-APOCHROMAT × 100 oil lens under an Axiovert 200M microscope (Carl Zeiss).
Ohashi A., Ohori M., Iwai K., Nakayama Y., Nambu T., Morishita D., Kawamoto T., Miyamoto M., Hirayama T., Okaniwa M., Banno H., Ishikawa T., Kandori H, & Iwata K. (2015). Aneuploidy generates proteotoxic stress and DNA damage concurrently with p53-mediated post-mitotic apoptosis in SAC-impaired cells. Nature Communications, 6, 7668.
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8

Immunoblotting Analysis of DNA Damage Response Proteins

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Immunoblotting was performed as previously reported [51 (link)]. Total proteins were extracted from the cultured cells. Samples containing 30-35 μg of total protein were subjected to 8-12% SDS polyacrylamide gel electrophoresis (PAGE), transferred onto a nitrocellulose membrane (Roche), and probed with following monoclonal primary antibodies: anti-actin (Sigma-Aldrich, Inc.), anti-TRF1, anti-hTERT, anti-hTERTTyr707, anti-γ-H2AX, anti-53BP1, anti-ATM, anti-p-ATM, anti-ATR, anti-Mer11, anti-Bcl-2, anti-Cyclin B1, anti-Cyclin D1, anti-aurora A, anti-p-aurora A, anti-p53 and anti-p21 (Santa Cruz Biotechnology, Inc.). HRP-conjugated goat anti-rabbit and goat anti-mouse antibody (ZSGB-BIO) were then used as secondary antibodies.
Cheng Y., Li Y., Ma C., Song Y., Xu H., Yu H., Xu S., Mu Q., Li H., Chen Y, & Zhao G. (2016). Arsenic trioxide inhibits glioma cell growth through induction of telomerase displacement and telomere dysfunction. Oncotarget, 7(11), 12682-12692.
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9

Western Blot Analysis of DNA Damage Markers

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Western blot analysis was performed as previously described (Cheung et al., 2011 (link)). Primary antibodies used include anti-WRN (Sigma-Aldrich, clone 195C), anti-γH2AX (Ser139) (Millipore, clone JBW301), anti-p53 (Santa Cruz Biotech, sc-126), anti-α-actinin (Santa Cruz Biotech, sc-17829), anti-p16 (Santa Cruz Biotech, sc-468), anti-p21 (Cell Signaling, #2946), anti-phospho-ATM (S1981) (Abcam, clone EP1890Y), anti-53BP1 (Santa Cruz Biotech, sc-22760), and anti-phospho-p53 (Ser15) (Cell Signaling, #9284).
Cheung H.H., Liu X., Canterel-Thouennon L., Li L., Edmonson C, & Rennert O.M. (2014). Telomerase Protects Werner Syndrome Lineage-Specific Stem Cells from Premature Aging. Stem Cell Reports, 2(4), 534-546.
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10

Quantifying DNA Damage Markers in NSCLC

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A549 NSCLC cells were incubated 30 min with SiBiGdNP at a concentration of 0.5 mM prior to irradiation. Cells were fixed in 4% paraformaldehyde in PBS at room temperature for 15 min. After fixation, cells were blocked in 1% BSA, 10% FBS, and 0.3% tritonX-100 in PBS. Next, cells were stained with anti γH2AX antibody (Millipore) and anti-53BP1 (Santa Cruz) overnight. Subsequently, cells were incubated with secondary anti-mouse AlexaFluor-594 conjugated IgG and anti-rabbit AlexaFluor-488 conjugated IgG (Abcam), respectively, for the noted primary antibodies. A semiquantitative analysis was performed to evaluate the number of foci per cell expressing the γH2Ax and 53BP1 markers. Images were visualized with an upright Carl Zeiss microscope with an HXP 120C light source and a 63x/1.4 oil plan-apochromat objective. Foci were identified in the images and their signal intensity was quantified using CellProfiler cell imaging software (version 2.1.1).39 (link)
Detappe A., Thomas E., Tibbitt M.W., Kunjachan S., Zavidij O., Parnandi N., Reznichenko E., Lux F., Tillement O, & Berbeco R. (2017). Ultrasmall Silica-Based Bismuth Gadolinium Nanoparticles for Dual Magnetic Resonance–Computed Tomography Image Guided Radiation Therapy. Nano letters, 17(3), 1733-1740.
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