Rnf168

Manufactured by Merck Group

Sourced in United Kingdom, Germany

RNF168 is a laboratory equipment product manufactured by Merck Group. It functions as a ubiquitin-protein ligase that catalyzes the addition of ubiquitin to its substrates, playing a role in the DNA damage response pathway.

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Lab products found in correlation

Rap80, by Fortis Life Sciences (2 mentions) Flag m2, by Merck Group (2 mentions) Ab15850, by Abcam (1 mentions) Ab18255, by Abcam (1 mentions) Ab6326, by Abcam (1 mentions) Rat anti brdu, by Abcam (1 mentions) Histone h2a, by Abcam (1 mentions) Vimentin, by Merck Group (1 mentions) Mouse anti brdu, by BD (1 mentions) Anti flag m2, by Merck Group (1 mentions) H3k9me2, by Merck Group (1 mentions) γh2ax jbw301, by Merck Group (1 mentions) Brca1 c20, by Santa Cruz Biotechnology (1 mentions) Enhanced chemiluminescence, by GE Healthcare (1 mentions) Horseradish peroxidase linked secondary antibody, by GE Healthcare (1 mentions) Nupage, by Thermo Fisher Scientific (1 mentions) α tubulin, by Merck Group (1 mentions) Rad51 h 92, by Santa Cruz Biotechnology (1 mentions) Fluorochrome conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions) Ha tag, by Santa Cruz Biotechnology (1 mentions)

Spelling variants of this product

RNF168 antibody (2 citations)

9 protocols using rnf168

Antibody Characterization Protocol for DNA Damage Response

Cited 1 time

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Antibodies used in this study included: UBR5 (#8755, CST^®, 1:1000); anti-mouse-BrdU (Becton Dickinson, different dilutions); anti-rat-BrdU (ab6326, Abcam; 1:500); PCNA-PC10 (Cancer Research, UK, 1:1000); Vimentin (V6389, Sigma-Aldrich, 1:1000); RNF8 (ab15850, Abcam; 1:2000); RNF168 (#ABE367, Millipore; 1:500); Tubulin (clone B-5-1-2, Sigma-Aldrich); Anti-Flag M2 (F3165, Sigma-Aldrich); 53BP1 (NB100-305, Novusbio, 1:2000); Chk1-PhosphoS345 (#2341 CST^®,1:1000); Chk1 (#2360 CST®,1:1000); Chk2-PhosphoT68 (#2661 CST^®, 1:500); Chk2(#2662 CST®, 1:1000); TRIP12 (ab86220, Abcam; 1:500); Histone H2A(ab18255, Abcam; 1:1000); polη (custom made, raised against the peptide: VQVEQRQNPHLRNKPC, 1:1000); polη-PhosphoS601(Eurogentec,(15 (link))); Histone H2A.X-PhosphoS139 (Upstate), RPA2 (1:1000, Millipore).

Cipolla L., Bertoletti F., Maffia A., Liang C.C., Lehmann A.R., Cohn M.A, & Sabbioneda S. (2019). UBR5 interacts with the replication fork and protects DNA replication from DNA polymerase η toxicity. Nucleic Acids Research, 47(21), 11268-11283.

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Antibody-based Analysis of DNA Damage Response

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The antibodies used against FANCJ (Bethyl Laboratories, Montgomery, TX, USA), BARD1 (BL518; Bethyl Laboratories), BRCA1 (C20; Santa Cruz Biotechnology, Santa Cruz, CA, USA), KAP‐pS824 (Bethyl Laboratories), RAP80, (Bethyl Laboratories), RNF168 (ABE367; Millipore, Darmstadt, Germany), and RAD51 (BioAcademia, Osaka, Japan) were rabbit polyclonal antibodies. The antibodies against CtIP (Active Motif, Carlsbad, CA, USA), conjugated ubiquitin (FK2; Nippon Bio‐Test Laboratories, Tokyo, Japan), HP1α (Millipore), HP1β (1MOD‐1A9; Millipore), HP1γ (2MOD‐1G6; Millipore), H3K9me2 (CMA307; Millipore), γH2AX (JBW301; Millipore), and α‐ and β‐tubulin (DMIA+BMIB; Neomarkers, Fremont, CA, USA) were mouse mAbs.

Wu W., Togashi Y., Johmura Y., Miyoshi Y., Nobuoka S., Nakanishi M, & Ohta T. (2016). HP1 regulates the localization of FANCJ at sites of DNA double‐strand breaks. Cancer Science, 107(10), 1406-1415.

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Antibody Characterization in Cell Culture

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All cells were grown in Dulbecco’s Eagle medium (DMEM) containing 10% Fetal Calf Serum (FCS), except B cells which were grown in RPMI-1640 supplemented with 15% FBS, 1% penicillin/streptomycin, 1% L-glutamine and 50 μM β-mercaptoethanol. Parental cells were tested for mycoplasma contamination.
Mouse antibodies employed were against Flag M2, α- and β-Tubulin (Sigma), γH2AX (Millipore) 6×His (Clontech), GFP (Cell signaling) and ATM (Santa Cruz), rabbit antibodies were against TopBP1, RIF1, PTIP, 53BP1 and phosphoKAP1 (S824) (All Bethyl Laboratories), 53BP1 (Santa Cruz), RNF168 (Millipore), TIRR (Sigma), phospho53BP1 (T543), phospho53BP1 (S25/29), HMGA1, H3 (All Cell Signaling) and AID (generated by the Chaudhuri Lab) and rat antibody against RPA2 (Cell Signaling). The RIF1 antibody used in immunofluorescence is a kind gift of Lifeng Xu (University of California, USA).

Drané P., Brault M.E., Cui G., Meghani K., Chaubey S., Detappe A., Parnandi N., He Y., Zheng X.F., Botuyan M.V., Kalousi A., Yewdell W.T., Münch C., Harper J.W., Chaudhuri J., Soutoglou E., Mer G, & Chowdhury D. (2017). TIRR regulates 53BP1 by masking its histone methyl-lysine binding function. Nature, 543(7644), 211-216.

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Western Blot Analysis of DNA Damage Signaling

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Whole cell extract (WCE) was resolved by SDS-PAGE (NuPAGE, Invitrogen) and transferred onto nitrocellulose membranes. Samples were incubated with primary antibodies recognizing RNF168 (Millipore), 53BP1 (Novus Biologicals), phospho-RPA2 (S4, S8), phospho-CHK1 (S317; all from Bethyl Laboratories), RPA2 (Cell Signaling Technologies) and α-tubulin (Sigma–Aldrich). Following incubation with the appropriate horseradish peroxidase-linked secondary antibodies (GE Healthcare), bands were visualized using enhanced chemiluminescence (GE Healthcare).

Zong D., Callén E., Pegoraro G., Lukas C., Lukas J, & Nussenzweig A. (2015). Ectopic expression of RNF168 and 53BP1 increases mutagenic but not physiological non-homologous end joining. Nucleic Acids Research, 43(10), 4950-4961.

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Immunofluorescence Analysis of DNA Damage Response

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For standard immunofluorescence studies, MEFs grown on coverslips were subjected to γ-irradiation (cesium-137) or treated with PARPi. At the indicated times post-treatment, cells were first pre-extracted (20 mM HEPES, pH 7.0, 50 mM NaCl, 3 mM MgCl₂, 0.3 M sucrose, 0.2% Triton X-100) for 5 min on ice to remove nucleoplasmic proteins and then sequentially fixed (4% paraformaldehyde), permeabilized (0.5% Triton X-100) and blocked (2% BSA). Samples were incubated with primary antibodies recognizing HA-tag (Santa Cruz Biotechnology), RNF168 (Millipore), conjugated ubiquitin (FK2, Sigma–Aldrich), 53BP1 (Novus Biologicals), RIF1 (14 (link)), RPA2 (Cell Signaling Technologies), RAD51 (H-92; Santa Cruz Biotechnology) followed by appropriate fluorochrome-conjugated secondary antibodies (Invitrogen). DNA was counterstained with DAPI. Foci images were captured with a fluorescence microscope at 63× magnification, unless otherwise stated. High-throughput automated imaging was used to captured chromatin-bound RPA at 20× magnification. Integrated nuclear RPA intensity was quantified using CellProfiler 2.0.

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Western Blot Analysis of DNA Damage Response Proteins

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Nuclear extracts were obtained using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific) and whole cell extracts were generated using RIPA buffer with protease and phosphatase inhibitors added. Proteins were separated by sodium dodecyl sulphate (SDS)-polyacrylamide gelelectrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane. Membranes were blocked with 5% nonfat milk in phosphate-buffered saline tween 20 (PBST) at room temperature for 1 h. Primary antibodies were incubated overnight at 4° and horseradish peroxidase (HRP)-conjugated secondary antibodies were incubated for 1 h at room temperature. The following primary antibodies were used: BRCA1 (EMD Millipore, catalog# OP92), RNF168 (EMD Millipore, #06-1130-I), Tubulin (Cell Signaling, catalog# 2148), GFP (Santa Cruz Biotechnology, catalog# sc-9996), RFP (ChromoTek, catalog# 6g6-20), FLAG (Cell Signaling, catalog# 14793 and Sigma Aldrich, catalog# F1804), phospho-Chk1 (Cell Signaling, catalog# 2344), Chk1 (Cell Signaling, catalog# 2360), POLD3 (Bethyl Laboratories, A301-244A), PALB2 (Bethyl Laboratories, catalog# A301-246A), BRCA2 (Bethyl Laboratories, catalog# A303-435A), RAD51 (Santa Cruz Biotechnology, catalog# sc-8349), RAD18 (Bethyl Laboratories, catalog# A301-340A), 53BP1 (Cell Signaling, catalog# 4908).

Krais J.J, & Johnson N. (2020). Ectopic RNF168 expression promotes break-induced replication-like DNA synthesis at stalled replication forks. Nucleic Acids Research, 48(8), 4298-4308.

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Immunofluorescence and Immunoblotting Antibodies

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The following antibodies were used for immunofluorescence and immunoblotting: RNF126 (mouse monoclonal antibody, Cat No. sc-376005, 1:500, Santa-Cruz Biotech, Santa Cruz, CA); FK2 (mouse monoclonal antibody, Cat No. 04-263, 1:250, EMD Millipore, Boston, MA); RNF8 (rabbit polyclonal antibody, Cat No. PABR-1160-042604, 1:500, Bethyl, Montgomery, TX); RNF168 (rabbit polyclonal antibody, Cat No. 06-1130, 1:1000, EMD Millipore); 53BP1 (rabbit polyclonal antibody, Cat No. A300-272A, 1:500, Bethyl); RAP80 (rabbit polyclonal antibody, Cat No. A300-763A, 1:250, Bethyl); γH2AX (mouse monoclonal antibody, Cat No. 05-636, 1:500, EMD Millipore); MDC1 [43] (link); HA (mouse monoclonal antibody, Cat No. M180-3, 1:5000, MBL, Nagoya, Japan); FLAG (mouse monoclonal antibody, Cat No. M185-3L, 1:5000, MBL); GFP (rabbit polyclonal antibody, Cat No. 598, 1:2000, MBL); β-actin (mouse monoclonal antibody, Cat No.66009-1-Ig, 1:5000, Proteintech, Chicago, IL). The following secondary antibodies were used: donkey anti-mouse IgG (Cat No.715-585-150, 1:1000, Jackson ImmunoResearch, West Grove, PA), donkey anti-rabbit IgG (Cat No. 711-585-152, 1:1000, Jackson ImmunoResearch).

Zhang L., Wang Z., Shi R., Zhu X., Zhou J., Peng B, & Xu X. (2018). RNF126 Quenches RNF168 Function in the DNA Damage Response. Genomics, Proteomics & Bioinformatics, 16(6), 428-438.

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Antibody Panel for DNA Damage Response

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Primary antibodies used in this study were UBA80 (Raybiotech, 144-02027-50), UBA52 (Bio-Rad, VPA00424), Flag M2 (Sigma, F1804), Myc (Santa Cruz, sc-40), GFP (Invitrogen, A11122), 53BP1 (Novus Biologicals, NB100-304), BRCA1 (Santa Cruz Biotechnology, SC-6954), γH2AX (Millipore, 05-636), H2AX (Cell Signaling, 2595S), H2A (Cell Signaling, 2578), tubulin (Abcam, ab6046), MDC1 (Abcam, ab11169), RNF168 (Sigma-Aldrich, ABE367), GST (Millipore, 71007-3), and MBP (Abcam, ab119994). For Western blotting, secondary antibodies—horseradish peroxidase-linked anti-rabbit immunoglobulin G and horseradish peroxidase-linked anti-mouse immunoglobulin G—were purchased from Cell Signaling (0704 and 0706). For immunofluorescence, Alexa Fluor 488 goat anti-rabbit and Alexa Fluor 594 goat anti-mouse antibodies were used (Invitrogen).

Lee S.O., Kelliher J.L., Song W., Tengler K., Sarkar A., Dray E, & Leung J.W. (2023). UBA80 and UBA52 fine-tune RNF168-dependent histone ubiquitination and DNA repair. The Journal of Biological Chemistry, 299(8), 105043.

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Western Blot Analysis of Irradiated Samples

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After irradiation, lysates were obtained at various time points and sonicated (QSonica) for 2 minutes in 0.4 M NaCl/20 mM HEPES buffer containing 1% Igepal, 0.1 mM ethylene glycol tetraacetic acid and ethylenediaminetetraacetic acid, 0.1 M dithiothreitol, 0.1 M phenylmethanesulfonylfluoride, and protease and phosphatase inhibitors (Sigma). Protein concentrations were measured with a DC protein assay kit (Bio-Rad, Hercules, CA, USA). Equalized proteins were subjected to electrophoresis at 60 mA in polyacrylamide pre-cast gels (Bio-Rad) and electrotransferred onto polyvinylidene fluoride membranes (Bio-Rad) at 4ºC for 1 hour at 100 V. Membranes were blocked with 5% milk (Bio-Rad). Antibodies were incubated overnight in 5% milk at various concentrations (P16, BD Bioscience, 1:5000; TRIP12, Santa Cruz, 1:1000; RNF168, Sigma, 1:1000; pRb, CST, 1:500; Rb, CST, 1:1000). Membranes were washed in Tris-buffered saline (Bio-Rad) with 0.1% Tween20 (Sigma) and incubated for 45 minutes at ambient temperature in 1:2000 anti-rabbit or anti-mouse IgG (GE Healthcare). Immunoreactions were visualized with the ECL2 system (Thermo Scientific) and then immediately exposed to autoradiographic film (Denville) for various periods. Images were scanned with an HP Scanjet 5550c and quantified with ImageJ software (http://rsbweb.nih.gov/ij/).

Wang L., Zhang P., Molkentine D.P., Chen C., Molkentine J.M., Piao H., Raju U., Zhang J., Valdecanas D.R., Tailor R.C., Thames H.D., Buchholz T.A., Chen J., Ma L., Mason K.A., Ang K.K., Meyn R.E, & Skinner H.D. (2016). TRIP12 as a mediator of human papillomavirus/p16-related radiation enhancement effects. Oncogene, 36(6), 820-828.

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