Chromatin was isolated from HEK293 cells. Cells were lysed in EBC-1 buffer (Tris pH 7.5, 100mM NaCl, 0.5% NP-40, 1mM EDTA), nuclear pellets were collected, washed in EBC-1 and resuspended in EBC-2 (Tris pH7.5, 300mM NaCl, 5mM CaCl2) for 30 minutes. The insoluble chromatin was pelleted and resuspended in EBC-2 for use in the release assay. This chromatin was incubated with the indicated concentration of compounds for 2 hrs at RT. The supernatant was collected and analyzed as the soluble fraction while the remaining pellet was resuspended in loading buffer, sonicated, and analyzed as the chromatin bound fraction. Anti-53BP1 (Sigma B4436) and anti-histone 3 (loading control) were used in western blots.
Anti 53bp1
Manufactured by Merck Group
Sourced in United States
Anti-53BP1 is a laboratory research tool that detects the 53BP1 protein, which is involved in DNA damage response pathways. It is used in experiments to study the function and localization of 53BP1 in cells. The product is intended for research use only and its performance characteristics have not been established for diagnostic or clinical purposes.
Automatically generated - may contain errors
Lab products found in correlation
Anti cyclin a, by Santa Cruz Biotechnology (3 mentions)
Anti p53, by Santa Cruz Biotechnology (2 mentions)
Anti ps10h3, by Santa Cruz Biotechnology (2 mentions)
Anti ps87mus81, by Abcepta (2 mentions)
Anti brdu, by BD (2 mentions)
Anti lamin b1, by Abcam (2 mentions)
Hrp conjugated matched secondary antibodies, by Jackson ImmunoResearch (2 mentions)
Anti flag, by Merck Group (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Anti ctsd, by Cell Signaling Technology (1 mentions)
Anti ran, by BD (1 mentions)
Anti ranbp1, by Santa Cruz Biotechnology (1 mentions)
Anti myo6, by Santa Cruz Biotechnology (1 mentions)
Anti γh2ax, by Cell Signaling Technology (1 mentions)
Anti mek, by Cell Signaling Technology (1 mentions)
Anti p21, by Santa Cruz Biotechnology (1 mentions)
Anti rb, by BD (1 mentions)
Anti caspase 3, by Cell Signaling Technology (1 mentions)
γh2ax, by Santa Cruz Biotechnology (1 mentions)
Specie specific fluorophore conjugated antibodies, by Thermo Fisher Scientific (1 mentions)
8 protocols using anti 53bp1
1
Chromatin Isolation and Compound Binding
2
Immunofluorescence Staining of Stress Granules and SOD1 Aggregates
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For immunofluorescence staining, classical protocols were used. Primary antibodies were incubated overnight at 4 °C, except for the γH2A.X antibody (2 h at room temperature). The following primary antibodies were used: to detect stress granules we used the antibody anti-EIF3ƞ (1:1000, Cat. No. sc-16377, Santa Cruz Biotechnology Inc., Dallas, TX, USA); natively folded SOD1 was detected with the anti-SOD1 (1:300, Cat. No. CB14379, Cell Applications Inc, San Diego, CA, USA) antibody; to detect aggregates of misfolded SOD1 we used an anti-SOD1 peptide sequence antibody raised against amino acid 58 to 72 (SOD1 aa 58–72, 1:500, Cat. No. AS13 2644, Agrisera, Vännäs, Sweden). To test the specificity of the peptide sequence antibody, we used a previously established HeLa cell model overexpressing a green fluorescence protein-tagged SOD1 mutant [46 (link)]. To detect DSBs we used anti-histone γH2A.X (1:500, Cat. No. 05-636, Sigma-Aldrich, St. Louis, MO, USA) and anti-53BP1 (1:1000, Cat. No. NB100-304, Novus Biologicals, Centennial, CO, USA). Nuclei were counter stained using Hoechst 33342 (Cat. No. 62249, ThermoFisher Scientific, Waltham, MA, USA).
3
Comprehensive Antibody Panel for Cellular Analysis
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We purchased anti-p53 (sc-126), anti-CRM1 (sc-5595), anti-RCC1 (sc-1162), anti-RanBP1 (sc-1160), anti-Myo6 (sc-393558), anti-EPLIN (sc-136399), and anti-HEXB (sc-376781) antibodies from Santa Cruz Biotechnology; anti-p21 (#554228), anti-Rb (#554136), anti-importin-β (#610560), and anti-Ran (#610340) antibodies from BD Biosciences; anti-p16 (#1963-1) and anti-RCC1 (#5134) from Epitomics; anti-pS10H3 (#9701), anti-γH2AX (#9718), anti-caspase-3 (#9665), anti-CALD1 (#12503), anti-CTSD (#2284), anti-MEK (#9122), anti-ADRM1 (#12019), anti-HSPD1 (#12165), and anti-HSP90B1 (#2104) from Cell Signaling Technology; anti-importin-α1 (NB100-1371) from Novus Biologicals; anti-actin (A1978) from Sigma; anti-53BP1 (#05-726) and anti-trimethylated-SPK (#07-1814) from Millipore; and anti-PSMB4 (AB137067), anti-NRMT (AB72660) and anti-LMNA (AB26300) from Abcam.
4
Quantification of DNA Damage Markers
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Cells were grown in 35-mm coverslips and harvested at the indicated times after treatments. For 53BP1 IF, after further washing with PBS, cells were fixed with 4% PFA at RT for 10 min. Cells were subsequently permeabilized with 0.4% Triton-X100. Staining with mouse polyclonal anti-53BP1 (1:300, Millipore), γ-H2AX (1:1000 Santa Cruz Biotechnology) or rabbit polyclonal anti-Cyclin A (1:100 Santacruz), pS10H3 (1:1000 Santa Cruz Biotechnology) diluted in a 1%BSA/0,1% saponin in PBS solution, was carried out for 1 h at RT. After extensive washing with PBS, specie-specific fluorophore-conjugated antibodies (Invitrogen) were applied for 1 h at RT followed by counterstaining with 0.5 mg/ml DAPI. Secondary antibodies were used at 1:200 dilution. Images were acquired as greyscale files using Metaview software (MDS Analytical Technologies) and processed using Adobe Photoshop CS3 (Adobe). For each time point, at least 200 nuclei were examined and foci were scored at 40×.
5
Western Blot Analysis of DNA Damage Markers
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Proteins were resolved by Mini Gel SDS-PAGE (Bio-Rad system) and transferred to nitrocellulose membrane (GE Healthcare) as previously described30 (link). All the blocking and antibody incubations were done in TBS −0.2% Tween-20 5% BSA (Fisher). The following primary antibodies were used: anti-53BP1 (1:1000, Millipore), anti-ATM (1:1000, Abcam), anti-Chk1-phS317 (1:1000, Cell Signalling Technology), anti-GFP (1:1000), anti-HA (1:1000, Sigma), anti-H2AX-P (1:1000, Abcam), anti-p53 (1:1000, DO-1, SantaCruz), anti-p53-phS15 (1:1000, NEB), anti-RNaseH1 (1:1000, Abcam), anti-TetR (1:1000, TETO2, MoBiTec), anti-tubulin (1:5000, Abcam), and appropriate HRP-conjugated secondary antibodies were used: anti-mouse (1:10000, Cell Signalling Technology), anti-rabbit (1:10000, Cell Signalling Technology) and anti-rat (1:10000, Abcam). Immuno-reactive bands were detected by chemoluminescence induced by Supersignal reagent and detected with the ImageQuant LAS 4000 machine (GE Healthcare). Quantification was performed using ImageJ.
6
Comprehensive Immunoblotting and Immunofluorescence Assay
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The primary antibodies used were: anti-MUS81 (1:1000; Santa Cruz Biotechnologies), anti-DDK (Flag Origene, WB 1:1000, IF 1:200), anti-cMYC (1:1000; Abcam), anti-CK2 (1:1000; Cell Signaling Technologies), anti-RxxpS/T (1:1000; Cell Signaling Technologies), anti-SLX4 (WB 1:1000, IF 1:200, Novus biologicals), anti-pS10H3 (1:1000, Santa Cruz Biotechnologies), anti-H3 (1:1000, Santa Cruz Biotechnologies), anti-EME1 (1:1000, Santa Cruz Biotechnologies), anti-EME2 (1:500, Invitrogen), anti-Cyclin A (WB: 1:1000, IF: 1:100, Santa Cruz Biotechnologies), anti 53BP1 (1:400, Millipore), anti-BrdU (1:50, Becton Dickinson), anti-pS139H2A.X (1:1000, Millipore), anti-γ-Tubulin (1:200, Sigma-Aldrich), anti-pS87MUS81 (WB 1:1000, IF 1:200, Abgent) and anti-Lamin B1 (1:10 000; Abcam). HRP-conjugated matched secondary antibodies were from Jackson Immunoresearch and were used at 1:40 000.
7
Immunofluorescence Staining of Cells
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On 6-well plates, sterilized coverslips were placed, then 2.5 × 105 cells were seeded on top of them. After growing overnight cells were exposed to different treatments. They were then washed gently with PBS 1x and fixed using 3% paraformaldehyde during 15 min at room temperature. Then, they were washed using PBS 1x and permeabilized with 0.25% Triton for 10 min. They were washed again with PBS 1x and blocked with BSA 2% during 1 h at room temperature. Samples were incubated with BSA 2% using anti-FLAG (Sigma Aldrich) or anti-53BP1 (Milipore) (1:100) for 45 min at 37 °C. A secondary antibody, anti-rabbit or anti-mouse, was incubated with BSA 2% for 20 min at 37 °C. Cells were washed with PBS 1x and then sealed with Vectashield antifade mounting medium + DAPI. Images were obtained using confocal microscopy (SP5 Confocal Leica Microscope). Detecting the anti-rabbit signal at 488 nm and the anti-mouse at 647 nm.
8
Immunostaining and Western Blot Antibodies
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The primary antibodies used were: anti-pS345CHK1 (1:100, Cell Signaling Technologies), anti-pThr1989ATR (1:100, GeneTex) anti-pS10H3 (1:1000, Santa Cruz Biotechnologies), anti-Cyclin A (IF: 1:100, Santa Cruz Biotechnologies), anti-53BP1 (1:300, Millipore), anti-BrdU (1:50, Becton Dickinson; anti-IdU detection), anti-pS87MUS81 (WB 1:1000, IF 1:200, Abgent), anti-RAD51 (WB 1:1000, IF 1:100 Bioss Antibodies), anti-α-Tubulin (1:50, Sigma-Aldrich), anti-Flag (1:1000, Sigma-Aldrich) and anti-Lamin B1 (1:10000, Abcam). HRP-conjugated matched secondary antibodies were from Jackson Immunoresearch and were used at 1:20 000.
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