LR and non-LR fractions from adipocytes and macrophages were obtained by Optiprep gradient centrifugation using a detergent-free protocol, as described previously (46 (link)). Briefly, cell pellets were homogenized in buffer (250 mmol/L sucrose, 1 mmol/L EDTA, 500 mmol/L sodium bicarbonate, pH 11). After centrifugation (1000g, 10 minutes), the postnuclear supernatant fraction was added to 60% Optiprep to a final concentration of 35% Optiprep, overlaid on a discontinuous gradient of 5%–35% Optiprep, and centrifuged at 326,512g in a Beckman NVT 65.2 rotor for 90 minutes at 4°C. Nine 0.5 mL fractions were collected and subjected to immunoblotting using antibodies against TLR4 (Cell Signaling Technology, 1:1000, 14358), NOX2 (Invitrogen, Thermo Fisher Scientific, 1:1000, PA5-76034), and caveolin-1 (Cell Signaling Technology, 1:1000, 3238).
Nvt 65.2 rotor
Manufactured by Beckman Coulter
The NVT 65.2 rotor is a fixed-angle rotor designed for use with Beckman Coulter ultracentrifuges. It is capable of accommodating sample volumes up to 13.5 mL and can achieve a maximum rotation speed of 65,000 rpm. The rotor's core function is to provide efficient separation and sedimentation of various biomolecules and cellular components during centrifugation processes.
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Lab products found in correlation
8 protocols using nvt 65.2 rotor
1
Lipid Raft Isolation from Adipocytes and Macrophages
2
Measurement of TOP1-DPCs by ICE Assay
3
Detecting Topoisomerase I-DNA Complexes
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TOP1-DPCs were isolated and detected using in vivo complex of enzyme (ICE) assay31 (link). Briefly, HCT116 cells were lysed in sarkosyl solution (1% w/v) after treatment. Cell lysates were sheared through a 25 g 5/8 needle (10 strokes) to reduce the viscosity of DNA and layered onto CsCl solution (150% w/v), followed by centrifugation in NVT 65.2 rotor (Beckman coulter) at 270,000 × g for 20 h at 25 °C. The resulting pellet containing nucleic acids and TOP1-DPCs was obtained and dissolved in TE buffer. The samples were quantitated and subjected to slot-blot for immunoblotting with various antibodies as indicated. Two μg of DNA is applied per sample. For mass spectrometric analysis, ICE samples were treated with RNase A to eliminate RNA contamination. Experiments were performed in triplicate and TOP1-DPCs were quantified by densitometric analysis using ImageJ.
4
Quantitative Analysis of TOP-DPCs
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TOP-DPCs were isolated and detected using ICE assay as previously described (37 (link)). Briefly, cells were lysed in sarkosyl solution [1% (w/v)] after treatment. Cell lysates were sheared through a 25-gauge 5/8 needle (10 strokes) to reduce the viscosity of DNA and layered onto CsCl solution [150% (w/v)], followed by centrifugation in NVT 65.2 rotor (Beckman coulter) at 42,000 rpm for 20 hours at 25°C. The resulting pellet containing nucleic acids and TOP-DPCs was obtained and dissolved in TE (Tris-EDTA) buffer. The samples were quantitated and subjected to slot-blot for immunoblotting with various antibodies as indicated. DNA (2 μg) was applied per sample. For mass spectrometric analysis, ICE samples were treated with ribonuclease A (RNase A) to eliminate RNA contamination. Experiments were performed in triplicate and TOP-DPCs were quantified by densitometric analysis using ImageJ.
5
Purification and Detection of TOP1-DPCs
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TOP1-DPCs were isolated and detected by ICE assay32 (link). Briefly, HEK293 cells were lysed in sarkosyl solution (1% w/v) then sheared with a 25-gauge 5/8 needle. The lysates were loaded onto CsCl solution (150% w/v) for ultracentrifugation in NVT 65.2 rotor (Beckman coulter) at 15,4893 × g for 20 h at 4 °C. The resulting DNA pellets were retrieved and suspended in TE buffer. The samples were quantitated and 2 μg DNA per sample was subjected to slot-blot using indicated antibodies.
6
Isolation and Detection of TOP2-DPCs
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Topoisomerase II DNA-protein complexes (TOP2-DPCs) were isolated and detected using in vivo complex of enzyme (ICE) assay as previously described (Anand et al., 2018 (link)). Briefly, 5 million cells were lysed in sarkosyl solution (1% w/v) after treatment. Cell lysates were sheared through a 25 g 5/8 needle (10 strokes) to reduce the viscosity of DNA and layered onto CsCl solution (150% w/v), followed by centrifugation in NVT 65.2 rotor (Beckman coulter) at 42,000 RPM for 20 hr at 25°C. The resulting pellet containing nucleic acids and TOP2-DPCs was obtained and dissolved in TE buffer. The samples were subjected to immunoblotting (slot blot) with anti-mouse TOP2β antibody (Novus). 2 μg of DNA is applied per sample. TOP2-DPCs were quantified by densitometric analysis using ImageJ.
7
Isolation and Detection of TOP1-DPCs
8
Quantification of Covalent DNA-Protein Crosslinks
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After treatments, yeast cells were pelleted and washed in 700 μl lysis buffer containing 6 M guanidinium thiocyanate, 1% sarkosyl, 4% Triton X 100, 10 mM Tris-HCl (pH 7.0), 20 mM EDTA, yeast protease inhibitor cocktail, 20 mM N-ethylmaleimide, and 1 mM DTT. Lysates were prepared as previously described (37 (link)) and ultracentrifuged for 18 hours at 42000 rpm in an NVT 65.2 rotor (Beckman coulter) at 25°C. Nucleic acids pellets containing TOP-DPCs were retrieved in ddH2O and digested with RNase A. Purified DNA samples were quantitated, and 10 μg of each sample was digested with micrococcal nuclease and subjected to SDS-PAGE for immunodetection of total TOP-DPCs, ubiquitylated TOP-DPCs, and SUMOylated TOP-DPCs. Each sample (2 μg) was subjected to slot-blot for immunoblotting with anti-dsDNA antibody to confirm equal amounts of DNA loading on the slot-blot.
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