Uv crosslinker

Manufactured by Hoefer

Sourced in United States

The UV Crosslinker is a laboratory equipment designed to expose samples to controlled doses of ultraviolet (UV) radiation. It is used to cross-link nucleic acids, proteins, and other biomolecules for various applications in molecular biology and biochemistry.

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Lab products found in correlation

Proteinase k, by Thermo Fisher Scientific (2 mentions) Trizol, by Thermo Fisher Scientific (2 mentions) Ab109255, by Abcam (1 mentions) Protein a and g agarose, by GE Healthcare (1 mentions) Mcf 7, by American Type Culture Collection (1 mentions) Quantity one software, by Bio-Rad (1 mentions) Phosphorimager screen, by Bio-Rad (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) Anti his tag antibody, by Cell Signaling Technology (1 mentions) Protein g dynabead, by Thermo Fisher Scientific (1 mentions) Formamide, by Thermo Fisher Scientific (1 mentions) Typhoon imager, by Cytiva (1 mentions) G box acquisition system, by Syngene (1 mentions) 3h uridine, by PerkinElmer (1 mentions) Imagequant tl image analysis software, by Cytiva (1 mentions) Tri reagent, by Merck Group (1 mentions) Dneasy plant mini kit, by Qiagen (1 mentions) Roti nylon membrane, by Carl Roth (1 mentions) Uvc 500, by Hoefer (1 mentions)

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UV crosslinker system (4 citations) Hoeffer UV Crosslinker (2 citations)

7 protocols using uv crosslinker

UV-CLIP Protocol for Studying IRF1 and IRF7 RNA Interactions

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UV-CLIP experiments were performed according to Schaukowitch et al.^{54 (link)}. Briefly, 1 × 10⁷ cells were cross-linked at 150 mJ/cm² using UV-CROSSLINKER (Hoefer Scientific Instruments, San Francisco, CA, USA) for 36 s in ice-cold PBS to preserve protein-RNA interaction. Totally, 10 µg rabbit anti-IRF1 (8478, Cell Signaling Technology Inc., Danvers, MA, USA) or rabbit anti-IRF7 (ab109255, Abcam, Cambridge, UK) antibodies were added to isolated lysed nuclei and incubated overnight at 4 °C with gentle rotation. IgG (Ab2410, Abcam, Cambridge, UK, rabbit) control sample was included. Bound RNAs were precipitated with protein A- and G-agarose (GE Healthcare Life Sciences, Buckinghamshire, UK), washed with increasingly stringent buffers as described in ref. ^{54 (link)} and eluted upon Proteinase K (Thermo Fisher Scientific Inc., Waltham, MA, USA) treatment for 2 h at 50 °C. Eluted RNA was isolated with an equal volume of phenol: chloroform:isoamyl 25:24:1 pH 8 (Thermo Fisher Scientific Inc., Waltham, MA, USA) and precipitated with ethanol 75% overnight. Isolated RNA was resuspended in nuclease-free water and qRT-PCR was performed as previously described.
RNA-Seq data of DROSHA and DGCR8 CLIP experiments were retrieved from GSE61979. Bedgraph files were then uploaded onto UCSC human assembly hg19 and visualized with UCSC Genome Browser.

Profumo V., Forte B., Percio S., Rotundo F., Doldi V., Ferrari E., Fenderico N., Dugo M., Romagnoli D., Benelli M., Valdagni R., Dolfini D., Zaffaroni N, & Gandellini P. (2019). LEADeR role of miR-205 host gene as long noncoding RNA in prostate basal cell differentiation. Nature Communications, 10, 307.

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Genotoxic Stress Response Assays

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Genotoxic stresses were induced by either radiation exposure or chemical treatments. For exposure of cells to UV irradiation, media was removed, washed two times with PBS, and then cells were exposed to UV radiation (10 mJ/m²) using a Hoefer Scientific UV cross-linker. For exposure of cells to ionizing radiation, cells were irradiated with 5 Gy using a Co⁶⁰ irradiator. Irradiated cells were then kept in the CO₂ incubator and collected at the indicated time periods. For H₂O₂, cells were grown in the press of 0.05% H₂O₂ for 2 h. Collected cells were then processed for either immunoblotting or immunoflourescence or comet assay or FACS analysis.

Islam S., Dutta P., Sahay O, & Santra M.K. (2021). β-TrCP1 facilitates cell cycle checkpoint activation, DNA repair, and cell survival through ablation of β-TrCP2 in response to genotoxic stress. The Journal of Biological Chemistry, 296, 100511.

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DNA Damage Assays in Human Cancer Cells

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Human cancer cell lines HeLa and MCF-7 were purchased from ATCC (Manassas, VA, USA). Cells were grown under standard tissue culture conditions (37 °C, 5% CO₂) in RPMI-1640 (HeLa) containing 10% fetal bovine serum or DMEM (MCF-7) containing 10% fetal bovine serum. For DNA damage treatment, cells were subjected to 300 ng/ml NCS or 10 Gy of ionizing radiation (γ-ray, generated from a Cobalt-60 source at the second affiliated hospital of Harbin Medical University, Harbin, China) or 20 J/m² UVC irradiation (UV Crosslinker; Hoefer Inc., Holliston, MA, USA).

Li Y., Sun H., Zhang C., Liu J., Zhang H., Fan F., Everley R.A., Ning X., Sun Y., Hu J., Liu J., Zhang J., Ye W., Qiu X., Dai S., Liu B., Xu H., Fu S., Gygi S.P, & Zhou C. (2017). Identification of translationally controlled tumor protein in promotion of DNA homologous recombination repair in cancer cells by affinity proteomics. Oncogene, 36(50), 6839-6849.

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Northern Blotting Analysis of RNA

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RNA (15 μg/sample) was electrophoresed on an agarose gel and transferred onto a nylon membrane for northern blotting analysis. Briefly, RNA (15 μg/sample) was denatured in 2.3× denaturant (2.5 mL 40× MOPS, 2.5 mL H₂O, 35 mL formaldehyde, 100 mL deionized formamide) for 15 min at 65 °C and separated by 1.2% agarose formamide gel in 1× MOPS buffer at 120 V. Electrophoresis was stopped when bromophenol blue dye reached 8 cm from wells. After electrophoresis, RNA samples were transferred on nylon positively charged membrane (GE Healthcare UK Limited, Little Chalfont, UK) with 20× SSC overnight and UV cross-linked onto membrane at 125 mJ in UV crosslinker (Hoefer, Holliston, MA, USA). For RNA detection, the blot was hybridized with a 32P-labeled probe [T3AG2] in Church buffer (0.5 M phosphate buffer pH 7.2, 7% SDS, 1 mM EDTA, 0.1% BSA) overnight at 55 °C. The gel was washed twice and exposed to a PhosphorImager screen and analyzed by Quantity One software (Biorad, Hercules, CA, USA).

Pompili L., Maresca C., Dello Stritto A., Biroccio A, & Salvati E. (2019). BRCA2 Deletion Induces Alternative Lengthening of Telomeres in Telomerase Positive Colon Cancer Cells. Genes, 10(9), 697.

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UV-Induced Riboproteomic Analysis

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Ten million HeLa cells were transiently transfected with mammalian expression vectors expressing His-DUSP14 or GFP as a control using Lipofectamine 2000 (Thermo Fisher) according to the manufacturer’s instructions. The next day, the cells were washed with PBS and irradiated once on ice with 150 mJ/cm² UV light (254 nm) in ice-cold PBS using a Hoefer Scientific UV Crosslinker. Cells were then pelleted and lysed in 1 ml of lysis buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1% Igepal CA-630, 0.1% SDS, 0.5% sodium deoxycholate, supplemented with protease inhibitors), sonicated, cleared, and adjusted to a protein concentration of 1 mg/ml. RNA was then partially digested with 0.2 U/ml RNase I for 3 min before immunoprecipitation with 10 μL of anti-His-tag antibody (Cell Signaling) pre-conjugated to 50 μL protein G Dynabeads (Thermo Fisher) for 1 h. The beads were then washed 5 times with lysis buffer before the bound RNAs were eluted off by Proteinase K (Thermo Fisher) digestion for 1 h at 65 °C. The RNA was then extracted using TRIzol (Thermo Fisher) and subjected to qPCR analysis with qPCR primers against JUNI, MALAT1, and PVT1.

Kumar V., Sabaté-Cadenas X., Soni I., Stern E., Vias C., Ginsberg D., Romá-Mateo C., Pulido R., Dodel M., Mardakheh F.K., Shkumatava A, & Shaulian E. (2024). The lincRNA JUNI regulates the stress-dependent induction of c-Jun, cellular migration and survival through the modulation of the DUSP14-JNK axis. Oncogene, 43(21), 1608-1619.

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Determination of rRNA Synthesis Rates

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The rate of rRNA synthesis was determined by metabolic labelling immediately before cell harvesting. 10 μCi [³H]-uridine (PerkinElmer) was added per 1ml of medium and cell cultures incubated for a further 30min to 3h as indicated. RNA was recovered with 1 ml Trizol (Invitrogen) according to the manufacturer’s protocol and resuspended in Formamide (Invitrogen). One microgram of RNA was loaded onto a 1% formaldehyde/MOPS Buffer gel [44 (link),45 (link)] or a 1% formaldehyde/TT Buffer gel [46 (link)]. The EtBr-stained gels were photographed using the G:BOX acquisition system (Syngene), irradiated in a UV cross-linker (Hoefer) for 5 min at maximum energy, and transferred to a Biodyne B membrane (Pall). The membrane was UV cross-linked at 70 J/cm², washed in water, air dried and exposed to a Phosphor BAS-IP TR 2025 E Tritium Screen (Cytiva). The screen was then analyzed using a Typhoon imager (Cytiva) and quantified using the ImageQuant TL image analysis software.

Tremblay M.G., Sibai D.S., Valère M., Mars J.C., Lessard F., Hori R.T., Khan M.M., Stefanovsky V.Y., LeDoux M.S, & Moss T. (2022). Ribosomal DNA promoter recognition is determined in vivo by cooperation between UBTF1 and SL1 and is compromised in the UBTF-E210K neuroregression syndrome. PLoS Genetics, 18(2), e1009644.

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Nucleic Acid Isolation and Analysis

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For isolation of nucleic acids, cells were harvested by centrifugation at 1,100 x g, 4°C for 6 min. Genomic DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to manufacturer's protocol or CTAB buffer (2% cetyltrimethylammonium bromide, 100 mM Tris-HCl, pH 8) followed by phenol/chloroform/isoamyl alcohol extraction (25:24:1, Carl Roth GmbH, Mannheim, Germany). DNAs were digested by restriction enzymes and separated on 0.8% agarose gels set up in TPE-buffer (89 mM Tris-phosphate, 2 mM Na 2 EDTA). Total cellular RNA was extracted by using the TRI reagent (Sigma-Aldrich, Saint Louis, USA), according to the manufacturer's instructions. RNAs were separated on 1% denaturing formaldehyde agarose gels. After separation nucleic acids were transferred to Roti Nylon + membrane (Roth, Karlsruhe, Germany), followed by UV light cross-linking (UV Crosslinker, UVC 500, Hoefer Inc., San Francisco, USA). Diglabeled probes were synthesized by PCR from total DNA or cloned cDNA (psbA) by using primers denoted in Supplemental Table S2. Hybridizations and detection of diglabeled probes were performed using standard methods.

Wang F., Dischinger K., Westrich L.D., Meindl I., Egidi F., Trösch R., Sommer F., Johnson X., Schroda M., Nickelsen J., Willmund F., Vallon O, & Bohne A.V. (2023). One-helix protein 2 is not required for the synthesis of photosystem II subunit D1 in Chlamydomonas. Plant physiology, 191(3).

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