SHEP cells were transfected using JetPrime (PolyPlus). When indicated, cells were treated with Ivermectin (Sigma I8898) 10 μM 2 h before collection. After 4 h, the medium was replaced with medium without serum. 24 h after transfection, cells were harvested, and nuclei were isolated from cytoplasm using the Nuclei Pure Prep Isolation kit (Sigma Aldrich). Input, cytoplasmic, and nuclear fractions were analyzed by western blot, with GAPDH as cytoplasmic marker and Histone H3 as nuclear marker. HEK293T cells were transfected with TrkC-KF-GFP and Hey1 using JetPrime. After 4 h, the medium was replaced with medium without serum. Twenty-four h after transfection, cells were harvested, and cytoplasmic, DNA-bound, and DNA-unbound fractions were separated using the Subcellular Protein Fractionation Kit for Cultured Cells (ThermoFisher Scientific). Fractions were analyzed by WB, using Histone as nuclear marker and actin as loading control.
Nuclei pure prep isolation kit
Manufactured by Merck Group
Sourced in Japan, United Kingdom
The Nuclei Pure Prep Isolation Kit is a product designed to isolate nuclei from various cell types. It provides a simple and efficient method to extract and purify nuclei for downstream applications such as genomic analysis, epigenetic studies, and nuclear proteomics.
Automatically generated - may contain errors
Lab products found in correlation
Ivermectin, by Merck Group (1 mentions)
Subcellular protein fractionation kit for cultured cells, by Thermo Fisher Scientific (1 mentions)
Jetprime, by Polyplus Transfection (1 mentions)
Optima xpn 80, by Beckman Coulter (1 mentions)
Hoechest 33342, by Thermo Fisher Scientific (1 mentions)
Mitotracker, by Thermo Fisher Scientific (1 mentions)
Itraq dissolution buffer, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Trypsin, by Promega (1 mentions)
Phosstop and complete ultra protease inhibitor tablets, by Roche (1 mentions)
5 protocols using nuclei pure prep isolation kit
1
Subcellular Fractionation and Analysis of Protein Expression
2
Isolation of Functional Cell Nuclei
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Functional nuclear isolation was performed using commercially available nuclei isolation kit (Nuclei Pure Prep Isolation Kit; Sigma-Aldrich Japan) as reported previously [6 (link)]. In brief, adherent cells were washed with D-PBS and scraped from the plate in the presence of lysis buffer. Cells in lysis media were carefully placed on the top of a 1.8 M sucrose gradient and the resulting suspension was centrifuged at 30,000×g for 45 min in a precooled swinging bucket ultracentrifuge (Optima XPN-80; Beckman Coulter Inc.CA, USA). Nuclei at the bottom of the centrifuge were washed with buffer provided with the kit. Purity of nuclei was assessed by Hoechst® 33342 and MitoTracker imaging and western blot using anti-OXPHOS, anti-GAPDH, and anti-total histone antibodies. For functional experiments, isolated nuclei were used immediately.
3
Isolation and Characterization of Functional Nuclei
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Functional nuclear isolation was performed using commercially available nuclei isolation kit (Nuclei Pure Prep Isolation Kit; Sigma‐Aldrich) as reported previously [15 (link)]. Purity of nuclei was assessed by western blot using anti‐OXPHOS, anti‐GAPDH, total histone antibodies, and imaging with nuclear marker Hoechest33342 (Thermo Fisher Scientific Inc.) and mitochondrial marker (MitoTracker; Thermo Fisher Scientific Inc.). For functional experiments, isolated nuclei were used immediately.
4
Isolation and Proteome Analysis of Hippocampal Nuclei
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Hippocampal nuclear extracts were prepared with the use of a Nuclei Pure Prep isolation kit (Sigma). Briefly, hippocampi were homogenized in 1 ml of lysis buffer containing 1 M DTT, 10% Triton X-100, protease inhibitor cocktail, and protein phosphatase inhibitor cocktails I and II (Sigma). 2 ml of 1.8 M sucrose was mixed into the homogenates and layered on top of 1 ml of a 1.8 M sucrose cushion. Samples were then centrifuged at 30,000 g for 45 min to pellet the nuclei. The supernatant was removed and the nuclei was resuspended in 300 μl of nuclei lysis buffer and sonicated for 2 min on ice. 1 μl of benzonase was added and incubated for 30 min at 37C. Samples were then centrifuged at 13,000 g for 10 min and the supernatant containing nuclear lysates was transferred.
100 μg of protein was used for iTRAQ experiments. Each sample was precipitated with ice-cold acetone, resuspended in iTRAQ dissolution buffer, and cysteine-blocked according to the manufacturer’s instrucitons (Applied Biosystems). Samples were then digested overnight with trypsin (Promega) at 37C (1:13 enzyme:substrate), differentially labeled with iTRAQ reagents, and combined as per protocol. The peptide samples were then separated by strong cationic exchange (SCX) chromatography and enriched using reversed-phase (RP) HPLC as described previously [42 (link)].
100 μg of protein was used for iTRAQ experiments. Each sample was precipitated with ice-cold acetone, resuspended in iTRAQ dissolution buffer, and cysteine-blocked according to the manufacturer’s instrucitons (Applied Biosystems). Samples were then digested overnight with trypsin (Promega) at 37C (1:13 enzyme:substrate), differentially labeled with iTRAQ reagents, and combined as per protocol. The peptide samples were then separated by strong cationic exchange (SCX) chromatography and enriched using reversed-phase (RP) HPLC as described previously [42 (link)].
5
Nuclear Protein Extraction and Purification
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Cells were washed twice in ice-cold PBS and then lysed in DET buffer (150 mM NaCl, 25 mM Hepes pH 7.5, 1 mM β-Mercaptoethanol, 0.2 mM CaCl 2 , 0.5 mM MgCl 2 , 0.5% NP-40) supplemented with PhosStop and Complete Ultra Protease Inhibitor tablets (Roche, Welwyn Garden City, UK). Lysates were incubated on ice for 10 minutes and centrifuged. The resulting pellets were washed twice in DET buffer, resuspended in RIPA buffer and cleared by centrifugation. For mass spectrometry, nuclear lysates were prepared using Nuclei PURE prep isolation kit (Sigma, Gillingham, UK).
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