Benzonase nuclease hc

Manufactured by Merck Group

Sourced in Switzerland

Benzonase Nuclease HC is an endonuclease enzyme that catalyzes the hydrolytic cleavage of DNA and RNA into oligonucleotides. It is used in various laboratory applications, including the purification of proteins and nucleic acids, cell lysis, and the removal of nucleic acid contaminants.

Automatically generated - may contain errors

Lab products found in correlation

Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions) Phosstop, by Roche (2 mentions) Lys c, by Promega (1 mentions) Bca assay, by Thermo Fisher Scientific (1 mentions) Nupage novex 4 12 bis tris protein gel, by Thermo Fisher Scientific (1 mentions) Iblot, by Thermo Fisher Scientific (1 mentions) Sc 15335, by Santa Cruz Biotechnology (1 mentions) Odyssey blocking buffer, by LI COR (1 mentions) Mgatp, by Merck Group (1 mentions) Insulin, by Merck Group (1 mentions) Nc nitrocellulose membranes, by Cytiva (1 mentions) Hydroxylamine solution, by Merck Group (1 mentions) Puromycin, by GoldBio (1 mentions) Sodium dodecyl sulfate (sds), by Bio-Rad (1 mentions) Lys c, by Fujifilm (1 mentions) Sep pak c18 cartridge, by Waters Corporation (1 mentions) High ph reversed phase peptide fractionation kit, by Thermo Fisher Scientific (1 mentions) Protein assay dye reagent concentrate, by Bio-Rad (1 mentions) Tmtpro 16plex label reagent, by Thermo Fisher Scientific (1 mentions) Phosstop, by Merck Group (1 mentions)

Close spelling variants of this product

Benzonase nuclease Benzonase HC Benzonase

11 protocols using benzonase nuclease hc

Protein Sample Preparation for Mass Spectrometry

Check if the same lab product or an alternative is used in the 5 most similar protocols

Nuclease enzyme
(Millipore Benzonase Nuclease HC; 71206-3) was added to cell lysates
to breakdown interfering DNA before quantification of protein concentration
by the BCA assay (Thermo Scientific 23225) according to the manufacturer’s
instruction. Disulfide bonds in the lysates were reduced with 15 mM
dithiothreitol (DTT) for 1 h at 37 °C, followed by alkylation
with 55 mM iodoacetamide (IAA) for 1 h at room temperature in the
dark. To remove urea and other substances that could interfere with
digestion, lysates were precipitated using chilled 50:50 ethanol:acetone
overnight at −20 °C. The resulting protein pellets were
then resuspended in 100 mM HEPES (pH 8.5) and sonicated for a total
of 15 min (30 s cycles) to break up the pellets. Proteins were digested
in a two-step digestion process with the 100:1 protein/protease ratio
of Lys-C (Promega; V1671) at 37 °C for 4 h, followed by 100:1
trypsin digestion (Promega; V5111) at 37 °C overnight.

Smith T.S., Andrejeva A., Christopher J., Crook O.M., Elzek M, & Lilley K.S. (2022). Prior Signal Acquisition Software Versions for Orbitrap Underestimate Low Isobaric Mass Tag Intensities, Without Detriment to Differential Abundance Experiments. ACS Measurement Science Au, 2(3), 233-240.

+ Open protocol

+ Expand

Extracting and Quantifying Proteins by Western Blot

Check if the same lab product or an alternative is used in the 5 most similar protocols

Total proteins were extracted with RIPA lysis buffer (89900, Thermo Scientific) supplemented with 1X Protease Inhibitor—Complete ULTRA tablets mini (5892791001, Roche) and 1X Benzonase nuclease HC (712063, Millipore) for 1 h at 4°C. Equal amounts of total cellular proteins were resolved on NuPAGE® Novex® 4–12% Bis-Tris Protein Gels (NP0336BOX, Thermo Fisher Scientific) and transferred to nitrocellulose membranes (iBlot, Thermo Fisher Scientific) following the manufacturer’s instructions. Membranes were then blocked in Odyssey Blocking Buffer (927-4-0000, Li-Cor) for 1 h at room temperature. Incubation with primary antibodies was carried out at 4°C overnight in Odyssey Blocking Buffer. The following antibodies were used: rabbit anti-β-SG (dilution 1/100, HPA011422, Sigma) and rabbit anti-Actinin alpha (H-300) (dilution 1/1000, sc-15335, Santa Cruz). After 1 h incubation with donkey anti-rabbit 680 antibody (926–68073, EuroBio) at room temperature, proteins were detected by fluorescence (Odyssey, Li-Cor) following the manufacturer’s instructions. Western blot signal quantification was performed with the Plot Lanes Analysis tool of Image J software (NIH).

Henriques S.F., Patissier C., Bourg N., Fecchio C., Sandona D., Marsolier J, & Richard I. (2018). Different outcome of sarcoglycan missense mutation between human and mouse. PLoS ONE, 13(1), e0191274.

+ Open protocol

+ Expand

Cryo-ET Analysis of Vimentin-Deficient MEFs

Check if the same lab product or an alternative is used in the 5 most similar protocols

For cryo-ET analysis, Vimentin^−/− MEFs were grown to ~80% confluency on EM-grids coated with a carbon mesh (R2/1, Au 200 mesh; Quantifoil, Jena, Germany). Using fine tweezers, EM grids with a homogenous distribution of cells were picked and washed in PBS/2 mM MgCl₂ for 5 sec. These grids were then incubated in pre-permeabilization buffer (PBS containing 0.1% Triton-X 100, 10 mM MgCl₂, 600 mM KCl and protease inhibitors) for 20–40 sec and subsequently rinsed in PBS/2 mM MgCl₂ for 10 sec. Next, the EM-grids were subjected to nuclease treatment using Benzonase (2.5 units/μl in PBS/2 mM MgCl₂; Millipore, Benzonase^® Nuclease HC, Purity > 99%) for 30 min at RT. The EM-grids were washed in PBS/2 mM MgCl₂ prior to addition of 3 μl of 10 nm or 15 nm fiducial gold marker (Aurion), diluted 1:500 from the original stock. Lastly, the EM-grids were manually blotted for ~3 sec from the reverse side and plunge frozen in liquid ethane.

Turgay Y., Eibauer M., Goldman A.E., Shimi T., Khayat M., Ben-Harush K., Dubrovsky-Gaupp A., Sapra K.T., Goldman R.D, & Medalia O. (2017). The molecular architecture of lamins in somatic cells. Nature, 543(7644), 261-264.

+ Open protocol

+ Expand

Hydrolysis of Styrene-Maleic Anhydride Copolymer

Check if the same lab product or an alternative is used in the 5 most similar protocols

SMA2000 (a copolymer of styrene and maleic anhydride) was supplied by Cray Valley and hydrolysed to form SMA using the protocol described by^{37 (link)}. Insulin, Zinc, Human, Recombinant, P. pastoris (#407709), Adenosine 5’-triphosphate magnesium salt from bacterial source (Mg-ATP) (#A9187) and ECLTM Select Western Blotting Detection Reagent (#GERPN2235) was supplied by Sigma Aldrich. NC Nitrocellulose Membranes (#15249794, Cytiva Amersham™ Protran™). Gibco™ 10 × concentrated Dulbecco’s phosphate-buffered saline (#14200-067), HaltTM Protease & Phosphatase Single-use Inhibitor Cocktail (100x) (#78442) and HaltTM Protease Single-use Inhibitor Cocktail (100x) (#78430) were supplied by ThermoFisher Scientific. Benzonase® Nuclease HC, Purity > 90% (#71205, Millipore). Calf intestine alkaline phosphatase Quick CIP (#M0525, New England BioLabs). Unless otherwise stated all chemical reagents and cell culture media and supplement were from Sigma Aldrich.

Morrison K.A., Wood L., Edler K.J., Doutch J., Price G.J., Koumanov F, & Whitley P. (2022). Membrane extraction with styrene-maleic acid copolymer results in insulin receptor autophosphorylation in the absence of ligand. Scientific Reports, 12, 3532.

+ Open protocol

+ Expand

Comprehensive Proteomics Workflow

Check if the same lab product or an alternative is used in the 5 most similar protocols

The following were used: FluoroBrite Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher, A1896701), benzonase nuclease HC (Millipore, 71205-3), urea (Sigma, catalogue number U5378), sodium dodecyl sulfate (Bio-Rad, catalogue number 1610302), high-glucose and high-pyruvate DMEM (Gibco/Invitrogen, 11995), low-glucose DMEM without amino acids (US Biological, D9800-13), TCEP (Gold Biotechnology), puromycin (Gold Biotechnology, P-600-100), protease inhibitor cocktail (Sigma-Aldrich, P8340), PhosSTOP (Sigma-Aldrich, 4906845001), trypsin (Promega, V511C), LysC (Wako Chemicals, 129-02541), EPPS (Sigma-Aldrich, catalogue number E9502), 2-chloroacetamide (Sigma-Aldrich, C0267), TMT 11plex Label Reagent (Thermo Fisher, catalogue numbers 90406 and A34807), TMTpro 16plex Label Reagent (Thermo Fisher, catalogue number A44520), hydroxylamine solution (Sigma catalogue number 438227), Empore SPE Disks C18 (3M - Sigma-Aldrich catalogue number 66883-U), Sep-Pak C18 Cartridge (Waters catalogue numbers WAT054960 and WAT054925), SOLA HRP SPE Cartridge, 10 mg (Thermo Fisher, catalogue number 60109-001), High-pH Reversed-Phase Peptide Fractionation Kit (Thermo Fisher, catalogue number 84868), Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad, catalogue number 5000006) and EBSS (Sigma-Aldrich cataloge number E3024).

Hickey K.L., Swarup S., Smith I.R., Paoli J.C., Miguel Whelan E., Paulo J.A, & Harper J.W. (2023). Proteome census upon nutrient stress reveals Golgiphagy membrane receptors. Nature, 623(7985), 167-174.

+ Open protocol

+ Expand

PBMC Proliferation Assay with Viral Antigens

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cryopreserved PBMCs were thawed, washed and left overnight in RPMI 1640 (Gibco) media containing 5% human AB serum (PAN‐Biotech), 2 mM L‐Glutamine, 1% penicillin/streptomycin and 5 U/ml Benzonase Nuclease HC, Purity >99% (Millipore). The next day, viable cells were counted via trypan blue exclusion and plated in media containing 5% human AB serum (PAN‐Biotech), 2 mM L‐Glutamine and 1% penicillin/streptomycin at 375000 cells per well into 96‐well flat‐bottomed plate (Corning) in triplicate and stimulated at 0.25 μg/ml with Prot_N (N), Prot_S1 (S1), Prot_S (S2) (Miltenyi Biotec), as well as positive control (PHA 0.5 μg/ml) and negative control (DMSO). Plates were incubated for 48 h. Proliferation was detected using a BrdU Cell Proliferation Assay kit (Sigma) as per manufacturer instructions. In short, BrdU labelling solution was added to wells for the last 20 h of incubation, cells were then dried, fixed and incubated for 90 min with anti‐BrdU‐POD (peroxidase) monoclonal antibody. Next, substrate solution (Tetramethylbenzidine) was added and colour change halted after 5 min with 25 μl 1 M H₂SO₄. Plates were read in an ELISA reader at 450 nm. Data are shown as stimulation index (SI) as a fold change compared to DMSO control wells.

Nattrass R.G., Krafft L., Zjablovskaja P., Schuster M., Kasmapour B., Sarisoy C., Minich J., Bach E, & Streeck H. (2022). The effect of age on the magnitude and longevity of Th1‐directed CD4 T‐cell responses to SARS‐CoV‐2. Immunology, 166(3), 327-340.

+ Open protocol

+ Expand

Efficient Cell Lysis and Protein Extraction

Check if the same lab product or an alternative is used in the 5 most similar protocols

For the recovery experiment, cell pellets were thawed on ice and ice cold lysis buffer was added. The composition and final concentration for each component in the lysis buffer was as follows: 50 mM HEPES, 0.8% NP-40, 1.5 mM MgCl2, 1x protease inhibitors (Roche cOmplete EDTA-free Protease Inhibitor Cocktail, Ref#11873580001), 1x phosphatase inhibitors (Roche PhosSTOP, Ref#04906845001) and 0.25 U/µl benzonase (Millipore Benzonase Nuclease HC, Ref#71206-3). Concentrated stocks of the lysis buffer were always freshly prepared and added volumes were adjusted to the remaining PBS supernatant in the cell pellets. Lysates were kept on a shaker at +4°C for one hour for lysis and nucleic acid digestion by benzonase.
For 2D-TPP, cell lysis was conducted as for recovery experiment with the exception that the cells were lysed immediately after heat treatment.
Cell lysis for recovery experiment to measure total protein abundance was conducted the same way as described above with few exceptions. Instead of NP-40, 1% SDS was used and the incubation was conducted at RT to avoid SDS precipitation with the incubation time decreased to 30 minutes. Occasionally with SDS-lysis the chromatin digestion at the used conditions was incomplete and the incubation time and the amount of benzonase was increased.

Määttä T.A., Rettel M., Helm D., Stein F., & Savitski M.M. (2020). Aggregation and Disaggregation Features of the Human Proteome.

+ Open protocol

+ Expand

Protein Sample Preparation for Mass Spectrometry

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cell pellets were resuspended in 300 µL extraction buffer (4% SDS in 100 mM triethylammonium bicarbonate (TEAB), phosphatase inhibitors (PhosSTOP™, Roche)). Samples were boiled (15 min, 95 °C, 350 rpm) and sonicated for 30 cycles in a bath sonicator (Bioruptor® Pico bath sonicator, Diagenode, Belgium; 30 s on, 30 s off) followed by probe sonication for 50 s (20 s on, 5 s off). 2 µL Benzonase® nuclease HC (250 U/µL, Merck Millipore) was added and incubated for 30 min (37 °C, 750 rpm). Reversibly oxidized cysteines were reduced with 10 mM TCEP (45 min, 22 °C, 1,000 rpm) followed by alkylation of free thiols with 20 mM iodoacetamide (45 min, 22 °C, 1,000 rpm, in the dark). Proteins were quantified using the fluorometric EZQ TM assay (Thermo Fisher Scientific).

Brenes A.J., Griesser E., Sinclair L.V., Davidson L.S.P., Prescott A.R., Singh F., Hogg E., Espejo-Serrano C., Jiang H., Yoshikawa H., Platani M., Swedlow J.R., Findlay G.M., Cantrell D.A., & Lamond A.I. (2021). Proteomic and functional comparison between human induced and embryonic stem cells.

+ Open protocol

+ Expand

Purification of Soluble m^sol Cgi-58 Protein

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells expressing m^solCgi-58 were disrupted by sonication in wash buffer (20 mm Tris-HCl, 500 mm NaCl, 30 mm imidazole, 0.1% Nonidet P-40, 3.5 mm β-mercaptoethanol, pH 7.8), supplemented with protease inhibitor mixture (Complete, EDTA-free Tabs-Roche, Roche Diagnostics, Basel, Switzerland), 750 units benzonase® nuclease HC (Merck, Darmstadt, Germany), and 1 mg/ml lysozyme. After centrifugation at 30,000 × g for 40 min, m^solCgi-58 was purified by affinity chromatography using the His-Trap FF column (GE Healthcare). The recombinant protein was eluted with a gradient of elution buffer (20 mm Tris-HCl, 500 mm NaCl, 250 mm imidazole, 10% glycerol, 3.5 mm β-mercaptoethanol, pH 7.8). After TEV cleavage (4 h at room temperature) m^solCgi-58 was further purified by gel filtration Superdex 200 (Sigma), in gel filtration buffer (15 mm Na₂HPO₄/KH₂PO₄, pH 7.8, 300 mm NaCl, 1 mm DTT, 1 mm EDTA). m^solCgi-58 samples were concentrated, and the buffer was exchanged to NMR titration buffer (15 mm Na₂HPO₄/KH₂PO₄, pH 7.0, 300 mm NaCl, 1 mm DTT, 1 mm EDTA).

Hofer P., Boeszoermenyi A., Jaeger D., Feiler U., Arthanari H., Mayer N., Zehender F., Rechberger G., Oberer M., Zimmermann R., Lass A., Haemmerle G., Breinbauer R., Zechner R, & Preiss-Landl K. (2015). Fatty Acid-binding Proteins Interact with Comparative Gene Identification-58 Linking Lipolysis with Lipid Ligand Shuttling. The Journal of Biological Chemistry, 290(30), 18438-18453.

+ Open protocol

+ Expand

Isolation of Mouse and Human T Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

OT-II, SMART-A1, or B6 mice were used for CD4⁺ expansions, and OT-I, 2 C, PMEL, and B6 mice were used for CD8⁺ expansions. Spleens and lymph nodes were harvested from 8 to 12-week-old mice and processed through a 70-μm cell strainer. Then, CD4⁺ and CD8⁺ T cells were isolated using corresponding no-touch isolation kits and magnetic columns from Miltenyi Biotech (Auburn, CA, USA) according to the manufacturer’s instructions.
For human isolations, blood was drawn from healthy donors per JHU IRB-approved protocols and PBMC were isolated by Ficoll-Paque PLUS (GE Healthcare) density gradient centrifugation. Cells were cryopreserved in a 90% FBS, 10% DMSO solution at 10⁷ cells/mL and stored in liquid nitrogen. Prior to use, cryopreserved PBMC were thawed with 50 U/mL benzonase Nuclease HC (EMD Millipore), washed, and then incubated overnight in a T cell culture medium at 37 °C. The following morning, CD4⁺ T cells were purified using no-touch CD4⁺ isolation kits and magnetic columns (Miltenyi).

Isser A., Silver A.B., Pruitt H.C., Mass M., Elias E.H., Aihara G., Kang S.S., Bachmann N., Chen Y.Y., Leonard E.K., Bieler J.G., Chaisawangwong W., Choy J., Shannon S.R., Gerecht S., Weber J.S., Spangler J.B, & Schneck J.P. (2022). Nanoparticle-based modulation of CD4+ T cell effector and helper functions enhances adoptive immunotherapy. Nature Communications, 13, 6086.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!