Cells on coverslips were fixed with 4% paraformaldehyde/2% Sucrose for 15 min, and triton extracted (0.5% Triton X-100 in PBS) for 4 min. Cells were blocked with 5%BSA/PBST and then incubated with respective antibodies for 30 min at 37 °C followed by incubation with secondary antibodies (FITC or Rhodamine) for 30 min at 37 °C. Primary antibodies used in immunofluorescence studies were BRCA1 (Upstate; 1:500), phospho-53BP1(S1778) (Cell Signaling, 2675S; 1:200), RPA (Cal Biochem, NA13; 1:100), 53BP1 (Bethyl Labs, A300-272A; 1:2,000), Rad51(Santa Cruz, SC-8349; 1:150), Mre11 (Genetex, GTX70212 1:200), CtIP (generous gift from Dr. Richard Baer), HA (Covance, MMS-101P; 1:500) and γ-H2AX (Millipore, 05-636; 1:5,000). For TPX2 (Bethyl Labs, A300-429A; 1:400) and γ-tubulin (Sigma- Aldrich, T6557; 1:1,000) staining, the cells were pre-fixed with acetone:methanol (3:7) at −20 °C for 10 min, followed by triton extraction (0.2% triton-X-100 in 20 mM HEPES, pH 7.4, 50 mM NaCl, 3 mM MgCl2, 300 mM Sucrose) at room temperature. Primary and secondary antibody staining was carried out as described above.
Phospho 53bp1 s1778
Manufactured by Cell Signaling Technology
Phospho-53BP1 S1778 is a laboratory reagent that detects the phosphorylation of the 53BP1 protein at serine residue 1778. 53BP1 is a key regulator of the DNA damage response pathway. Phosphorylation of 53BP1 at S1778 is involved in the cellular response to DNA double-strand breaks.
Automatically generated - may contain errors
Lab products found in correlation
Gtx70212, by GeneTex (1 mentions)
Mms 101p, by Merck Group (1 mentions)
T6557, by Merck Group (1 mentions)
Rad51, by Santa Cruz Biotechnology (1 mentions)
Imager 600, by Cytiva (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Precision red assay, by Cytoskeleton (1 mentions)
γh2ax, by Cell Signaling Technology (1 mentions)
Phospho atm s1981, by Cell Signaling Technology (1 mentions)
Fibrillarin, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 and 594, by Thermo Fisher Scientific (1 mentions)
Nis element, by Nikon (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
A1r confocal microscope, by Nikon (1 mentions)
3 protocols using phospho 53bp1 s1778
1
Immunofluorescence Staining of DNA Damage Repair Proteins
2
Western Blot Assay for DNA Damage Markers
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Cells were lysed in RIPA buffer (Millipore) containing HALT protease and phosphatase inhibitor cocktail (ThermoScientific) and sonicated to complete lysis. Lysates were clarified by centrifugation before protein concentrations were assayed using the Precision Red assay (Cytoskeleton). Equal masses were electrophoresed by SDS-PAGE and wet transferred to PVDF membranes (BioRad). When probing for proteins with molecular weights over 200 kDa, precast gradient gels were used (Invitrogen and BioRad) and wet transfers were done at 30 V for 16 h. Primary antibodies against γ-H2AX (Cell Signaling, 9718S), GLI1 (Cell Signaling, 2643S), fibrillarin (Cell Signaling, 2639S), I-SceI (Abcam, ab216263), 53BP1 (Cell Signaling, 4937S), phospho-53BP1 S1778 (Cell Signaling, 2675S), ATM (Cell Signaling, 2873S), phospho-ATM S1981 (Cell Signaling, 5883S), NBS1 (Cell Signaling, 3002S), phospho-NBS1 S343 (Cell Signaling, 3001S), and β-actin (Sigma) were used, as well as secondary HRP-conjugated antibodies against mouse and rabbit IgG (GE) when appropriate. Chemiluminescence images were captured using the Imager 600 (Amersham). Densitometry was calculated using ImageJ software (NIH).
3
Quantifying Nuclear Co-localization of Phospho-53BP1, GLI1, and UBF1
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SUM1315 cells incubated for 1 h at room temperature with 1:200 GLI1 (Cell Signaling, 2553S) and 1:200 phospho-53BP1 S1778 (Cell Signaling, 2675S) primary antibodies in 5% BSA. After washing, cells were incubated in 1:100 secondary anti-mouse and anti-rabbit IgG antibodies conjugated to Alexa Fluor 488 and 594 (Invitrogen) in 5% BSA, followed by three washes in PBS before incubation for 1 h with 1:50 UBF Antibody (F-9) conjugated to Alexa Fluor 647 (Santa Cruz, sc-13125 AF647). The cells were stained with DAPI (Fisher) and mounted with VECTASHIELD (Vector Laboratories) and analyzed using a Nikon A1R Confocal Microscope at 60X. Intensity of phospho-53BP1-GLI1-UBF1 co-localization was determined using the manufacturer's protocol for calculating binary intersection mean intensity in NIS-Elements (Nikon) software from five random confocal images.
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