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Anti 53bp1 antibody

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The Anti-53BP1 antibody is a laboratory reagent used to detect the 53BP1 protein, which plays a role in the cellular response to DNA double-strand breaks. This antibody can be utilized in various research applications, such as immunoblotting, immunoprecipitation, and immunofluorescence studies, to investigate the expression and localization of 53BP1 in biological samples.

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Lab products found in correlation

Minimum essential medium (mem), by Thermo Fisher Scientific (2 mentions) Human epidermal growth factor (hegf), by Thermo Fisher Scientific (2 mentions) Hydrocortisone, by Thermo Fisher Scientific (2 mentions) Insulin, by Thermo Fisher Scientific (2 mentions) Cholera toxin, by Thermo Fisher Scientific (2 mentions) Bovine pituitatry hormone, by Thermo Fisher Scientific (2 mentions) Fitc labeled antirabbit iggs, by Thermo Fisher Scientific (2 mentions) T red labeled antimouse iggs, by Thermo Fisher Scientific (2 mentions) 53bp1 rabbit antibody, by Fortis Life Sciences (1 mentions) Streptavidin beads, by Thermo Fisher Scientific (1 mentions) Alexa fluor 555, by Thermo Fisher Scientific (1 mentions) Alexa fluor 488 antibody, by Jackson ImmunoResearch (1 mentions) Vectashield with dapi, by Vector Laboratories (1 mentions) Clsm 700, by Zeiss (1 mentions) Mouse γh2ax antibody, by Merck Group (1 mentions) Alexa fluor 594 labeled anti rabbit igg secondary antibody, by Thermo Fisher Scientific (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions) Formaldehyde, by Fujifilm (1 mentions) Glycerol, by Fujifilm (1 mentions)

Spelling variants of this product

Anti-53BP1 (14 citations) 53BP1 antibody (6 citations) Anti-53BP1 rabbit polyclonal antibody (2 citations)

6 protocols using anti 53bp1 antibody

1

Quantifying DNA Damage in Breast Cancer Cells

Cited 2 times
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To determine DNA damage, immunofluorescence was performed with vehicle and CB-treated cells using 53BP1 rabbit antibody (#A300-272AT, Bethyl Laboratories). Breast cancer cells were grown on coverslips and treated vehicle or CB. The cells were then fixed with 4% cold paraformaldehyde followed by permeabilization and blocking using 5% normal goat serum in 0.1% Triton X-100 for 30 min at room temperature. The cells were then incubated with anti-53BP1 antibody (1:200 dilution; Bethyl Laboratories) and Phospho γ-H2AX (1:200 dilution; Ab2893) overnight followed by incubation with TRITC-conjugated goat anti-rabbit secondary antibody (1:500). The cells were counterstained with DAPI and observed under a fluorescence microscope15 (link), 40 (link).
Rajamanickam S., Park J.H., Subbarayalu P., Timilsina S., Bates K., Yadav P., Nirzhor S.S., Eedunuri V., Mohammad T.A., Jung K.H., Onyeagucha B., Abdelfattah N., Benevides R., Lee G., Chen Y., Vadlamudi R., Brenner A., Kaklamani V., Jatoi I., Kuhn J., Hromas R., Gupta Y.K., Kaipparettu B.A., Arbiser J.L, & Rao M.K. (2022). Targeting aberrant replication and DNA repair events for treating breast cancers. Communications Biology, 5, 493.
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2

Immunoprecipitation of 53BP1 Protein

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Cells were lysed with NTEN buffer (20mM Tris-HCl, pH 8.0, 100mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40) containing pepstatin A and aprotinin on ice for 30 minutes. Clear cell lysates were incubated with either protein A/G-agarose beads coupled with anti-53BP1 antibody (#A700–011, Bethyl) or streptavidin beads (#17511301, Fisher Scientific) for 3 hours at 4°C. Beads were then washed 4 times with NTEN buffer and boiled in 2xLaemmli buffer, and proteins were separated by SDS-PAGE. PVDF membranes were blocked in 5% milk in TBST buffer and then probed with antibodies as indicated.
Zhang F., Lou L., Peng B., Song X., Reizes O., Almasan A, & Gong Z. (2020). Nudix hydrolase NUDT16 regulates 53BP1 protein by reversing 53BP1 ADP-ribosylation. Cancer research, 80(5), 999-1010.
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3

Immunostaining of DNA Damage Markers

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Nonmalignant human mammary epithelial cells (MCF10A, ATCC) were cultivated in minimum essential medium (MEM, Invitrogen), supplemented with bovine pituitatry hormone (13 mg/mL), hydrocortisone (0.5 mg/mL), hEGF (10 µg/mL), insulin (5 mg/mL), and cholera toxin (100 ng/mL) (Invitrogen). Cell culture was performed in Permanox plastic 8-well Lab-Tek chamber slides (Nalge Nunc International Corporation) at 37C, with 95% humidity and 5% CO2. The cells were grown to a confluent layer prior to irradiation. After three washing steps with PBS, cells were blocked with 0.2% BSA for 15 min, incubated with the primary antibodies for 15 min, washed with PBS, and incubated with the secondary antibodies for 15 min. The primary antibodies were a rabbit polyclonal anti 53BP1 antibody (1:100 from 1 mg/mL, Bethyl Laboratories) and a mouse monoclonal to phosphohistone H2AX antibody (1:100 from 1 mg/mL, clone JBW301, Upstate Cell Signaling Solutions Inc.). The corresponding secondary antibodies were either FITC-labeled antirabbit IgGs, or T-Red-labeled antimouse IgGs (Molecular Probes Invitrogen). After a further washing step with PBS, the cells were counterstained with DAPI. All steps were performed at room temperature.
Pariset E., Bertucci A., Petay M., Malkani S., Macha A.L., Lima I.G., Gonzalez V.G., Tin A.S., Tang J., Plante I., Cekanaviciute E., Vazquez M, & Costes S.V. (2020). DNA Damage Baseline Predicts Resilience to Space Radiation and Radiotherapy. Cell reports, 33(10), 108434.
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4

Quantification of DNA Damage Markers

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TK6 cells (0.5 million/ml) were exposed to IPMS (750μM) for 24 hours. Cells were then fixed using 2% formaldehyde / PBS for 20 min, washed in PBS, and spotted on to double frosted microscopic glass slides (Fisher Scientific) at a concentration of 2 million/ml. Blocking was achieved using 5% (w/v) bovine serum albumin (BSA) in PBS-TT for 30 min. Cells were then incubated with 1:500 rabbit polyclonal anti-53BP1 antibody (Bethyl Laboratories, Montgomery, TX) and mouse γH2AX antibody (Millipore Corporation, CA, USA) in 1% BSA/PBS-TT for overnight at 4oC. Cells were then washed in PBS, incubated in 1:250 anti-mouse Alexa Fluor 555 (Life Technologies) and Alexa Fluor 488 antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) for 1h. After washing with PBS, slides were mounted with a cover slip using Vectashield with DAPI (Vector Laboratories) and sealed using nail polish. Slides were analyzed using confocal microscopy (Zeiss CLSM 700). Optical sections through the nuclei were captured at 0.5 μm intervals, and the images were obtained by maximum projection of the individual sections.
Hashimoto K., Sharma V., Sasanuma H., Tian X., Takata M., Takeda S., Swenberg J.A, & Nakamura J. (2016). Poor recognition of O6-isopropyl dG by MGMT triggers double strand break-mediated cell death and micronucleus induction in FANC-deficient cells. Oncotarget, 7(37), 59795-59808.
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5

Quantifying DNA Double-Strand Breaks

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To estimate the induction of DNA double-strand breaks immediately after irradiation, cells on 22 × 22 mm cover slips in 35 mm dishes were irradiated with 1 Gy of γ-rays. At 0.5, 2 or 24 h after irradiation, the cells were fixed with 4% formaldehyde (Wako) in phosphate-buffered saline (PBS). Then, the cells were permeabilized for 10 min in 0.5% Triton X-100 in PBS and washed with PBS. The cover slips were incubated with anti-53BP1 antibody (Bethyl Laboratories, TX, USA) in Tris-buffered saline supplemented with 5% skim milk (TBS-DT) for 2 h at 37°C. The primary antibody was washed with PBS, and an Alexa Fluor 594–labeled anti-rabbit IgG secondary antibody (Invitrogen Ltd, California, USA) and 4’,6-diamidino-2-phenylindole (DAPI; Invitrogen) were added. The cover slips were incubated for 1 h at 37°C and washed with PBS. Then, the cover slips were sealed onto glass slides with 0.03 ml PBS containing 10% glycerol (Wako). The cells were observed using a fluorescence microscope. The numbers of 53BP1 foci were counted on digitized images using Image J software.
Tamari Y., Kashino G, & Mori H. (2017). Acquisition of radioresistance by IL-6 treatment is caused by suppression of oxidative stress derived from mitochondria after γ-irradiation. Journal of Radiation Research, 58(4), 412-420.
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6

Immunostaining of DNA Damage Markers

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Nonmalignant human mammary epithelial cells (MCF10A, ATCC) were cultivated in minimum essential medium (MEM, Invitrogen), supplemented with bovine pituitatry hormone (13 mg/mL), hydrocortisone (0.5 mg/mL), hEGF (10 µg/mL), insulin (5 mg/mL), and cholera toxin (100 ng/mL) (Invitrogen). Cell culture was performed in Permanox plastic 8-well Lab-Tek chamber slides (Nalge Nunc International Corporation) at 37C, with 95% humidity and 5% CO2. The cells were grown to a confluent layer prior to irradiation. After three washing steps with PBS, cells were blocked with 0.2% BSA for 15 min, incubated with the primary antibodies for 15 min, washed with PBS, and incubated with the secondary antibodies for 15 min. The primary antibodies were a rabbit polyclonal anti 53BP1 antibody (1:100 from 1 mg/mL, Bethyl Laboratories) and a mouse monoclonal to phosphohistone H2AX antibody (1:100 from 1 mg/mL, clone JBW301, Upstate Cell Signaling Solutions Inc.). The corresponding secondary antibodies were either FITC-labeled antirabbit IgGs, or T-Red-labeled antimouse IgGs (Molecular Probes Invitrogen). After a further washing step with PBS, the cells were counterstained with DAPI. All steps were performed at room temperature.
Pariset E., Bertucci A., Petay M., Malkani S., Macha A.L., Lima I.G., Gonzalez V.G., Tin A.S., Tang J., Plante I., Cekanaviciute E., Vazquez M, & Costes S.V. (2020). DNA Damage Baseline Predicts Resilience to Space Radiation and Radiotherapy. Cell reports, 33(10), 108434.
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