Rad18

Manufactured by Fortis Life Sciences

Sourced in United States

The RAD18 is a laboratory equipment designed for specific scientific applications. It serves a core function within the research workflow, though its exact intended uses may vary. A detailed and unbiased description cannot be provided while maintaining the requested level of conciseness and objectivity.

Automatically generated - may contain errors

Lab products found in correlation

Rad51, by Santa Cruz Biotechnology (3 mentions) Fancd2, by Santa Cruz Biotechnology (3 mentions) Mycotest kit, by Thermo Fisher Scientific (3 mentions) γh2ax, by Merck Group (3 mentions) Brca2, by Fortis Life Sciences (2 mentions) Gapdh, by Santa Cruz Biotechnology (2 mentions) Anti mouse igg cy3, by Thermo Fisher Scientific (2 mentions) Tubulin, by Cell Signaling Technology (1 mentions) Palb2, by Fortis Life Sciences (1 mentions) Phospho chk1, by Cell Signaling Technology (1 mentions) Rnf168, by Merck Group (1 mentions) Pold3, by Fortis Life Sciences (1 mentions) Ne per nuclear and cytoplasmic extraction kit, by Thermo Fisher Scientific (1 mentions) Flag tag (flag), by Cell Signaling Technology (1 mentions) Penicillin streptomycin sulfate, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Omega Scientific (1 mentions) Anti mouse igg hrp, by Cytiva (1 mentions) Ecl anti rabbit igg, by Cytiva (1 mentions) Proliferating cell nuclear antigen (pcna), by Santa Cruz Biotechnology (1 mentions) Nitrocellulose membrane, by Bio-Rad (1 mentions)

5 protocols using rad18

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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Characterization of DNA Repair in Cancer Cell Lines

Cited 1 time

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H1299 (human non-small cell lung carcinoma) and A2780 (human ovarian cancer) were obtained from ATCC, and HCT116 and HCT116 RAD18^−/− (human colon cancer) cells were from Dr. Tadahiro Shiomi [27 (link)] and as used earlier [29 (link)]. Cells were cultured in Dulbecco's Modification of Eagle's Medium (DMEM) (Mediatech) supplemented with 10% fetal bovine serum (FBS) (Omega Scientific) and 1x Penicillin/Streptomycin sulfate (Gibco) [57 (link)]. Cells were routinely tested for mycoplasma contamination using Mycotest kit (Invitrogen) and were used prior to ten passages. CPT (Sigma) was used at concentrations and time periods indicated. Antibodies against the following targets were used: FANCD2, Rad51, 53BP1, GAPDH (all from Santa Cruz Biotechnology) BRCA2, RAD18 (Bethyl Laboratories) and γH2AX (Millipore). To express wild-type or the RING mutant RAD18, HCT116−/− cells were transfected with pcDNAmycRAD18 and pcDNARAD18 C28F, respectively.

Tripathi K., Mani C., Clark D.W, & Palle K. (2016). Rad18 is required for functional interactions between FANCD2, BRCA2, and Rad51 to repair DNA topoisomerase 1-poisons induced lesions and promote fork recovery. Oncotarget, 7(11), 12537-12553.

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Western Blot Protocol for Protein Analysis

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Protein samples were suspended in SDS Loading Buffer (50mM Tris-Cl pH6.8, 2% SDS, 0.1% bromophenol blue, 10% glycerol, 10% β-mercaptoethanol) and boiled for 5 minutes. Samples were run on 7.5% SDS-polyacrylamide gels and transferred to nitrocellulose membrane(Bio-Rad, Hercules, CA). Membranes were blocked in TBS-T(TBS+0.1% Tween-20) with 10% milk for 1.5 hours and incubated in primary antibody (FANCD2 1:2000, RAD51 1:2000, PCNA 1:500, Ku86 1:5000, Santa Cruz, and RAD18 1:1000, Bethyl) for one hour at room temperature in TBS-T+1% milk. After washing, membranes were incubated in secondary antibody(ECL anti-rabbit IgG or anti-mouse IgG-HRP, Amersham, Princeton, NY) 1:5000 in TBS-T+1% milk for 1 hour at room temperature. Blots were washed in TBS-T three times for 5 minutes each and developed by chemiluminescence (Supersignal West Pico Kit or Supersignal West Femto kit, Pierce, Rockford, IL). The quantification of Western blot was carried out using ImageJ software. We measured mean values of each band with fixed area. The relative values of each band were shown. For ub-PCNA we measured mean values of each band, and the values were then corrected with mean values of PCNA. The final values were shown.

Chen X., Bosques L., Sung P, & Kupfer G.M. (2015). A novel role for non-ubiquitinated FANCD2 in response to hydroxyurea induced DNA damage. Oncogene, 35(1), 22-34.

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NSCLC Cell Line Characterization

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A549 and H1299 cells (classified as NSCLC cells) were collected from American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in Dulbecco’s modified Eagle’s medium enriched with 10% fetal bovine serum and 100 U/mL penicillin and 100 μg/mL streptomycin sulfate. The possible mycoplasma contamination was checked by Mycotest kit (Invitrogen, Chicago, IL, USA). The following antibodies were used in the study: pATM, FANCD2, Chk1, β-Actin, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); Rad18 from Bethyl Laboratories, Inc. (Montgomery, TX, USA); pATM-Ser1981, pChk1-Ser317, pChk2, FANCJ, Bid, caspase-3, Snail and cyclin B are from Cell Signaling Technologies (Danvers, MA, USA); and γH2AX from Millipore company (Darmstadt, Germany); and E-cadherin from Becton Dickinson company (BD Biosciences, San Jose, CA, USA) Cell Signaling Technologies (The horseradish peroxidase (HRP)-conjugated secondary antibodies, such as anti-mouse IgG-Cy3 and anti-rabbit IgG, were procured from Molecular Probes (Santa Cruz, CA, USA).

Ahmed A.G., Hussein U.K., Ahmed A.E., Kim K.M., Mahmoud H.M., Hammouda O., Jang K.Y, & Bishayee A. (2020). Mustard Seed (Brassica nigra) Extract Exhibits Antiproliferative Effect against Human Lung Cancer Cells through Differential Regulation of Apoptosis, Cell Cycle, Migration, and Invasion. Molecules, 25(9), 2069.

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Isothiocyanate Modulation of DNA Damage Response

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The NSCLC cell lines A549 (human adenocarcinoma epithelial cell line) and H1299 (human were cultured in Dulbecco's modified Eagle's medium, supplemented with 10% FBS, 100 μg/ml streptomycin sulfate and 100 U/ml penicillin. Normal human bronchial epithelial cells were grown in BEGM™ Bronchial Epithelial Cell Growth Medium as described previously [51 (link)]. Cells were routinely tested for mycoplasma contamination using Mycotest kit (Invitrogen) and cells within 10 passages were used in the experiments. AITC and PITC (Sigma, St. Louis, MO) stock solutions were prepared by dissolving in anhydrous DMSO and stored at −20°C. These stock solutions were further diluted to required concentration before adding to the cells. Antibodies to the following antigens used in this study include: ATR, ATM, Chk1, FANCD2 and GAPDH were from Santa Cruz Biotechnology, Inc.; Rad18, from Bethyl Laboratories, Inc.; phospho-ATM-Ser1981, phospho-Chk1 Ser-317 were Cell Signaling Technologies, γH2AX is from Millipore. The secondary antibodies like anti-mouse IgG-Cy3, anti-mouse IgG-FITC, anti-rabbit IgG-FITC were from Molecular Probes.

Tripathi K., Hussein U.K., Anupalli R., Barnett R., Bachaboina L., Scalici J., Rocconi R.P., Owen L.B., Piazza G.A, & Palle K. (2015). Allyl isothiocyanate induces replication-associated DNA damage response in NSCLC cells and sensitizes to ionizing radiation. Oncotarget, 6(7), 5237-5252.

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