Benzonase

Manufactured by Thermo Fisher Scientific

Sourced in United States

Benzonase is a recombinant, genetically engineered endonuclease that non-specifically degrades DNA and RNA. It has a high catalytic activity and can efficiently remove nucleic acids from samples.

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Lab products found in correlation

33 protocols using benzonase

Cryopreservation and Thawing of Human PBMCs

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The total PBMCs were counted, and 3 × 10⁶ cells were placed into each cryo-vial tube along with 0.5 ml of media A. Then, 0.5 ml of media B (20% DMSO in RPMI 1640 medium, Sigma) was added to each cryo-vial tube. The cryo-vial tubes were then sealed and placed into a cell freezing container containing isopropanol. The cells were kept at −80°C for 24 h and then put into a liquid nitrogen (LN2) canister with LN2.
When thawing frozen PBMCs, the frozen cells were quickly thawed at 37°C for 1 min. Cells were resuspended in RPMI 1640 complete medium with benzonase (25 U/ml) (Sigma). The PBMCs were then centrifuged twice at 300 g for 8 min. Finally, the cells were resuspended in 1 ml of complete RPMI 1640 medium (Gibco) without benzonase for counting, and the cell concentration was adjusted with complete RPMI 1640 medium without benzonase for the flow cytometry assay.

Lu Y., Xu W., Gu Y., Chang X., Wei G., Rong Z., Qin L., Chen X, & Zhou F. (2019). Non-small Cell Lung Cancer Cells Modulate the Development of Human CD1c+ Conventional Dendritic Cell Subsets Mediated by CD103 and CD205. Frontiers in Immunology, 10, 2829.

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Protein Elution and Precipitation

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To elute proteins, beads were rinsed with benzonase elution buffer (20 mM Tris pH 8.5, 2 mM MgCl₂, 0.05% N-laroylsarcosine (NLS), 0.5 mM TCEP) while magnetically separated and incubated with 500 U benzonase (Merck Millipore, 71206-3) and 44 U RNase I (Thermo Fisher Scientific, AM2239) in benzonase elution buffer at 37°C and 1000 rpm overnight. The supernatant was separated from the beads and transferred into new tubes. To precipitate proteins, 100% Trichloroacetic acid (TCA) was added to a final concentration of 20%, followed by incubation on ice for 2 h and centrifugation at 14,000 g, 4°C for 1 h. The pellet washed once in ice-cold acetone, air-dried and dissolved in 8 M urea in 50 mM Tris pH 8.5.

Schmidt N., Ganskih S., Wei Y., Gabel A., Zielinski S., Keshishian H., Lareau C.A., Zimmermann L., Makroczyova J., Pearce C., Krey K., Hennig T., Stegmaier S., Moyon L., Horlacher M., Werner S., Aydin J., Olguin-Nava M., Potabattula R., Kibe A., Dölken L., Smyth R.P., Caliskan N., Marsico A., Krempl C., Bodem J., Pichlmair A., Carr S.A., Chlanda P., Erhard F, & Munschauer M. (2023). SND1 binds SARS-CoV-2 negative-sense RNA and promotes viral RNA synthesis through NSP9. Cell, 186(22), 4834-4850.e23.

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Quantification of Mitochondrial Protein Synthesis

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Mitochondrial protein synthesis was analyzed by metabolic labelling with ³⁵S methionine/cysteine (Richter et al, 2015 (link)). The cells were pretreated with anisomycin (100 μg/ml) to inhibit cytoplasmic translation then pulsed with 200 μCi/ml ³⁵S Met–Cys (EasyTag-Perkin Elmer). In chase experiments, the cells were pulsed for 30 min with radiolabel then the medium removed and replaced with fresh medium lacking the radioisotope for the indicated time. Equal amounts of sample protein were first treated with Benzonase (Thermo Fisher Scientific) on ice and then resuspended in 1× translation loading buffer (186 mM Tris-Cl, pH 6.7, 15% glycerol, 2% SDS, 0.5 mg/ml bromophenol blue, and 6% β-mercaptoethanol). 12%–20% gradient Tris-glycine SDS–PAGE gels were used to separate the samples and then dried for exposure with a phosphoscreen and scanned with a Typhoon 9400 or Typhoon FLA 7000 (GE Healthcare) for quantification. The gels were rehydrated in water and Coomassie stained to confirm loading.

Richter U., Ng K.Y., Suomi F., Marttinen P., Turunen T., Jackson C., Suomalainen A., Vihinen H., Jokitalo E., Nyman T.A., Isokallio M.A., Stewart J.B., Mancini C., Brusco A., Seneca S., Lombès A., Taylor R.W, & Battersby B.J. (2019). Mitochondrial stress response triggered by defects in protein synthesis quality control. Life Science Alliance, 2(1), e201800219.

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Western Blot Protein Analysis

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Cells were collected with trypsin, quickly washed in PBS, counted with Cellometer Auto T4 (Nexcelom Bioscience) and directly lysed in 4× NuPage LDS sample buffer at 10,000 cells per µl. PARPi and PARGi were added to 4× NuPage LDS sample buffer to inhibit PARP and PARG activity in solution, except for the westerns shown in Fig. 3d. Proteins were gently homogenized using a Benzonase (ThermoFisher), denatured for 10 min at 70 °C and resolved by SDS–PAGE electrophoresis, transferred to nitrocellulose membranes, blocked in 5% milk in TBST for 30 min and probed. HRP-linked antirabbit or mouse (Amersham) was used for secondary antibodies, and the HRP signal was visualized with SuperSignal ECL substrate (Pierce) as per the manufacturer’s instructions. For additional antibody information, see Supplementary Table 1.

Wondisford A.R., Lee J., Lu R., Schuller M., Groslambert J., Bhargava R., Schamus-Haynes S., Cespedes L.C., Opresko P.L., Pickett H.A., Min J., Ahel I, & O’Sullivan R.J. (2024). Deregulated DNA ADP-ribosylation impairs telomere replication. Nature Structural & Molecular Biology, 31(5), 791-800.

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Western Blot Analysis of Cellular Proteins

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Whole cell extracts from cell lines were obtained using RIPA buffer (Thermofisher) containing a protease inhibitor cocktail (Roche) and benzonase (Thermofisher). Protein concentrations were quantified using a BCA gold assay (Thermofisher). Equal amounts of total protein were prepared in 4X NuPAGE LDS Sample Buffer (NP0007, Invitrogen) and 5% BME, and boiled at 95°C for 5 min. The protein samples were resolved on 4–12% NuPAGE Bis-Tris gel with protein molecular weight standards (PageRuler Prestained Protein Ladder, 10 to 180 kDa, Thermofisher). The gels were transferred onto PVDF membranes using the Trans-Blot Turbo Transfer System (Bio-Rad). Primary antibodies used in this study included: rabbit anti-c-Myc antibody [Y69] (ab32072, Abcam, 1:1000), mouse anti-β-Actin antibody (3700, Cell Signaling, 1:5000). Fluorescent secondary antibodies used in this study were IRDye 680RD Donkey anti-Mouse IgG Secondary Antibody and IRDye 800CW Donkey anti-Rabbit IgG Secondary Antibody (Licor, 1:5000).

Chen J., Zheng Q., Hicks J.L., Trabzonlu L., Ozbek B., Jones T., Vaghasia A., Larman T.C., Wang R., Markowski M.C., Denmeade S.R., Pienta K.J., Hruban R.H., Antonarakis E.S., Gupta A., Dang C.V., Yegnasubramanian S, & De Marzo A.M. (2023). MYC-driven increases in mitochondrial DNA copy number occur early and persist throughout prostatic cancer progression. bioRxiv.

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Affinity Purification and Mass Spectrometry of FLAG-RBM22

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One to 2 × 10⁸ HepG2 cells expressing inducible FLAG-RBM22 were induced for 48 h by Dox (100 ng/ml final concentration) or H₂O (control). Cells were harvested by centrifugation at 500 g for 6 min at 4°C. Cell pellets were suspended in cell lysis buffer (50 mM Tris HCl, pH 7.4, 50 mM NaCl, 1 mM EDTA, 1% TritonX-100, and proteinase inhibitor cocktail) containing 200 units/ml Benzonase (Thermo, 88,701). After rotating for 30 min at 4°C, the cell lysates were cleared by spinning at 13,000 rpm for 30 min at 4°C. The supernatants were incubated with ANTI-FLAG M2 Affinity Gel (Sigma-Aldrich, A2220) overnight at 4°C with rotation. Beads were washed four times with IP wash buffer (20 mM Tris–HCl pH 7.5, 150 mM NaCl, 1.5 mM MgCl₂, 3 mM EDTA, 10% (v/v) glycerol, 0.1% (v/v) NP-40) and once with TE buffer (10 mM Tris–HCl pH 8.0, 1 mM EDTA). The immunoprecipitations were eluted using HRV 3C protease by incubation for 2 h at 22°C and subjected to label-free quantitative mass spectrometry.

Du X., Qin W., Yang C., Dai L., San M., Xia Y., Zhou S., Wang M., Wu S., Zhang S., Zhou H., Li F., He F., Tang J., Chen J.Y., Zhou Y, & Xiao R. (2024). RBM22 regulates RNA polymerase II 5′ pausing, elongation rate, and termination by coordinating 7SK-P-TEFb complex and SPT5. Genome Biology, 25, 102.

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Stimulation of PBMCs from TB Patients

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PBMCs from 5 patients with ATB, 5 with LTB and five HCs were thawed at 37°C followed by addition of 1 mL RPMI-1640 media supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin (P/S) and 250 U/mL Benzonase (all from ThermoFisher). The cells were washed twice (300g for 5 min) in media followed by resuspension in RPMI-1640 culture media supplemented with 10% FBS, 1% P/S, 0.3 g/L L-Glutamine and 25 mM HEPES and counted. The cells were then plated in 24-well plates at 2x10⁶ PBMCs/mL in culture media containing either 5 μg/mL PHA, 10 μg/mL PPD, or 25 μg/mL LAM or PIM, or left untreated (PBS), for 24 h in a 37°C 5% CO₂ incubator. 4 h before collection, 5 μg/mL of brefeldin A and 2 μM Monensin (both ThermoFisher) were added to each well. PHA was used as positive control for PBMCs responsiveness (Supplemental Figures 1, 2). The choice of concentration of LAM and PIM was based on titrations with cytokine secretion into supernatants as read-out (data not shown). The 24 h stimulation did not alter cell numbers between the conditions (Supplemental Figure 3).

Silva C.S., Sundling C., Folkesson E., Fröberg G., Nobrega C., Canto-Gomes J., Chambers B.J., Lakshmikanth T., Brodin P., Bruchfeld J., Nigou J., Correia-Neves M, & Källenius G. (2021). High Dimensional Immune Profiling Reveals Different Response Patterns in Active and Latent Tuberculosis Following Stimulation With Mycobacterial Glycolipids. Frontiers in Immunology, 12, 727300.

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Thermal Proteome Profiling of Cellular Samples

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TPP was performed as previously described with slight modifications (30 (link)). Briefly, cells were harvested through trypsinization, pelleted at 300g for 5 min at 4 °C, and washed with ice-cold PBS. The supernatant was removed, and cells were resuspended in 1.1 ml of ice-cold PBS. Then, 100-μl aliquots of the cell suspension were transferred to a PCR tube and centrifuged at 300g for 5 min at 4 °C. Next, 80 μl of the supernatant was removed, and each sample was heated at a certain temperature within a defined temperature gradient (37.0–66.1 °C) for 3 min followed by incubation at room temperature for 3 min. Heated cell pellets were resuspended in 130 μl ice-cold lysis buffer (0.5% NP-40, 1 mM MgCl₂, complete mini EDTA free protease inhibitors (Roches), 250 U/ml benzonase (Thermo) in PBS) through pipetting, and cells were lysed at 4 °C for 1 h. The lysates were transferred to a 0.45-μm 96-well filter plate (Millipore, prewetted with the lysis buffer), and the filter plate was transferred to an extraction plate vacuum manifold (Promega). Then, the sample was filtered to remove protein aggregates. Next, 100 μl of each sample was transferred to a new 96-well plate and kept at −80 °C until further prepared for MS analysis.

Yin K, & Wu R. (2023). Investigation of Cellular Response to the HSP90 Inhibition in Human Cells Through Thermal Proteome Profiling. Molecular & Cellular Proteomics : MCP, 22(6), 100560.

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Mitochondrial Protein Synthesis Analysis

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Mitochondrial protein synthesis was analyzed by metabolic labeling with ³⁵S methionine/cysteine^{44 (link)}. Cells were pre-treated with anisomycin (100 μg/ml) to inhibit cytoplasmic translation then pulsed with 200 μCi/ml ³⁵S Met-Cys (EasyTag-Perkin Elmer). In chase experiments, cells were pulsed for 30 min with radiolabel then the medium removed and replaced with fresh medium lacking the radioisotope for the indicated time. Equal amounts of sample protein were first treated with Benzonase (Thermofisher) on ice and then resuspended in 1x translation loading buffer (186 mM Tris-Cl pH 6.7, 15% glycerol, 2% SDS, 0.5 mg/ml bromophenol blue, 6% beta-mercaptoethanol). A 12–20% gradient Tris-Glycine SDS-PAGE was used to separate samples then dried for exposure with a Phospho screen and scanned with a Typhoon 9400 or Typhoon FLA 7000 (GE Healthcare) for quantification. Gels were rehydrated in water and Coomassie stained to confirm loading.

Richter U., Evans M.E., Clark W.C., Marttinen P., Shoubridge E.A., Suomalainen A., Wredenberg A., Wedell A., Pan T, & Battersby B.J. (2018). RNA modification landscape of the human mitochondrial tRNALys regulates protein synthesis. Nature Communications, 9, 3966.

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Isolation of Nuclear Envelopes

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Nuclear envelopes were prepared by the method described by Dreger et al. (42 (link)). Briefly, the nuclei (5–8 mg of protein) were suspended in 25 ml of ice-cold TP buffer (10 mM Tris–HCl, pH 8.0, 10 mM Na₂HPO₄, 1 mM PMSF, 1 mM benzamidine) containing 250 μg/ml of heparin, 1 mM Na₃VO₄, 10 mM NaF and 400 units of benzonase (ThermoFisher Scientific). The suspension was stirred for 90 min at 4°C. Nuclear envelopes were then pelleted by centrifugation at 10 000 × g for 30 min at 4°C, and resuspended in STM 0.25 buffer (50 mM Tris–HCl, pH 7.5, 0.25 M sucrose, 5 mM MgCl₂, 2 mM DTT, 1 mM PMSF, 1 mM benzamidine) at ∼0.2–0.5 mg/ml.

Sanchez-Lopez I., Orantos-Aguilera Y., Pozo-Guisado E., Alvarez-Barrientos A., Lilla S., Zanivan S., Lachaud C, & Martin-Romero F.J. (2024). STIM1 translocation to the nucleus protects cells from DNA damage. Nucleic Acids Research, 52(5), 2389-2415.

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