Tissues from mice or cells were solubilized in 2 mL of 1% Triton X-100 (Junsei Chemical Co., Ltd.) in MES-buffered saline (MBS, 25 mM MES, pH 6.5, 150 mM NaCl). After homogenizing with 10 up-and-down strokes of a tight-fitting Dounce homogenizer, the tissue or cellular extracts were adjusted to 4 mL with sucrose concentrations of 45% or 40%, respectively, and overlaid with 4 mL of 30% sucrose and 4 mL of 5% sucrose in MBS. The sucrose gradient was formed by centrifugation at 200,000 × g for 18–20 h at 4 °C using a Beckman SW41ti rotor. After centrifugation, the sucrose gradients were fractionated into 12 fractions without pelleting, and an opaque buoyant band corresponding to the LRs was collected at the interface between the 30% and 5% sucrose gradients. The same quantity of proteins from each fraction was used for immunoblotting analysis.
Triton x 100
Manufactured by Junsei
Sourced in Japan
Triton X-100 is a non-ionic detergent commonly used in biochemical and cell biology applications. It is a clear, colorless liquid with a viscosity similar to water. Triton X-100 is used to solubilize and extract membrane proteins, disrupt cell membranes, and lyse cells. It is also employed in various assays and purification procedures.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 700, by Zeiss (3 mentions)
Blocking one histo, by Nacalai Tesque (2 mentions)
Sw41ti rotor, by Beckman Coulter (1 mentions)
Tripalmitin, by Merck Group (1 mentions)
Tricaprin, by Merck Group (1 mentions)
Trilaurin, by Merck Group (1 mentions)
Tributyrin, by Merck Group (1 mentions)
Triolein, by Merck Group (1 mentions)
Hitrap q sepharose fast flow, by Cytiva (1 mentions)
Ammonium sulfate, by Junsei (1 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
P nitrophenyl palmitate p npp, by Merck Group (1 mentions)
Trizma base, by Merck Group (1 mentions)
Diethyl ether, by Merck Group (1 mentions)
Sodium chloride (nacl), by Junsei (1 mentions)
Potassium chloride (kcl), by Duksan Pure Chemicals (1 mentions)
Oleic acid, by Duksan Pure Chemicals (1 mentions)
Slowfade diamond antifade mountant with 4 6 diamidino 2 phenylindole dapi, by Thermo Fisher Scientific (1 mentions)
D1s7w, by Cell Signaling Technology (1 mentions)
Alexa fluor 488 secondary antibody, by Thermo Fisher Scientific (1 mentions)
5 protocols using triton x 100
1
Lipid Raft Isolation and Fractionation
2
Isolation and Characterization of Chestnut Lipase
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Korean chestnuts (Castanea crenata) were purchased from local markets. They were peeled, sealed in a vacuum plastic bag, and stored at 4 °C for subsequent use. Before use, the chestnuts were covered with a layer of wet cotton and allowed to germinate for 3 days at 18 °C.
HiTrap DEAE Sepharose Fast Flow, HiTrap Q Sepharose Fast Flow, and HiPrep Sephacryl S-100 HR were purchased from Cytiva (Uppsala, Sweden). Trizma® base (≥ 99.9%), diethyl ether (≥ 99.9%), p-nitrophenyl palmitate (p-NPP), sodium dodecyl sulfate (SDS), isooctane (IOT), tributyrin, tricaproin, tricaprin, trilaurin, tripalmitin, and triolein (≥ 99.0%) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Ammonium sulfate (≥ 99.5%), Triton X-100, sodium chloride (≥ 99.5%), and agar were purchased from Junsei Chemical Co., Ltd. (Tokyo, Japan). Boric acid, potassium chloride, and oleic acid were obtained from Duksan Pure Chemicals Co., Ltd. (Ansan, Gyeonggi-do, Korea). The Costar® 96-well microplates (clear wall, clear bottom) used for the fluorometric assay were purchased from Corning Co. (Corning, NY, USA). All other chemicals were of analytical grade and were used without further purification.
HiTrap DEAE Sepharose Fast Flow, HiTrap Q Sepharose Fast Flow, and HiPrep Sephacryl S-100 HR were purchased from Cytiva (Uppsala, Sweden). Trizma® base (≥ 99.9%), diethyl ether (≥ 99.9%), p-nitrophenyl palmitate (p-NPP), sodium dodecyl sulfate (SDS), isooctane (IOT), tributyrin, tricaproin, tricaprin, trilaurin, tripalmitin, and triolein (≥ 99.0%) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Ammonium sulfate (≥ 99.5%), Triton X-100, sodium chloride (≥ 99.5%), and agar were purchased from Junsei Chemical Co., Ltd. (Tokyo, Japan). Boric acid, potassium chloride, and oleic acid were obtained from Duksan Pure Chemicals Co., Ltd. (Ansan, Gyeonggi-do, Korea). The Costar® 96-well microplates (clear wall, clear bottom) used for the fluorometric assay were purchased from Corning Co. (Corning, NY, USA). All other chemicals were of analytical grade and were used without further purification.
3
Assessing Apoptosis via DAPI Staining
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The morphological changes in apoptotic cells were assessed by fluorescent microscopy following DAPI staining. Briefly, HepG2 cells were seeded at a density of 5 × 105 cells/2 ml in 6-well plates, then grown with four different concentrations of GEGR for 24 h in a 37°C incubator. After washing once with ×1 PBS, cells were incubated with 5 mM H2O2 for another 12 h, then fixed in 4% formaldehyde (Cat. No. 69360-0380, Junsei Chemical Co. Ltd., Tokyo, Japan) for 1 h and permeated with 0.1% Triton X-100 (Cat. No. T1020, Biosesang Inc., Seongnam, Korea) for 5 min. Next, the DNA-specific fluorochrome DAPI (100 μM, Cat. No. D1306, Invitrogen) was applied to each well, after which samples were incubated for 10 min in the dark at room temperature. Finally, the cells were washed three times with ×1 PBS and examined using a fluorescent microscope (Olympus IX71, Tokyo, Japan) at ×400 magnification.
4
Visualizing HIF-1α Nuclear Translocation
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GMSCs (3 × 104 cells/well) were seeded onto glass coverslips and stimulated with the indicated amounts of TNF-α and IFN-α for 6 and 24 h to observe the nuclear accumulation of HIF-1α. The cells were washed twice with PBS (pH 7.4), fixed with 4% paraformaldehyde for 20 min, blocked with Blocking One Histo (Nacalai Tesque) for 5 min at room temperature, treated with 0.5% Triton X-100 (Junsei, Tokyo, Japan), which penetrated into the cells, for 10 min at room temperature, and stained with primary anti-HIF-1α (1:200, D1S7W, Cell Signaling Technology) antibody for 1 h at room temperature. Immunofluorescent labelling of HIF-1α was performed using anti-HIF-1α (1:200, D1S7W, Cell Signaling Technology). Subsequently, Alexa Fluor® 488 secondary antibody (1:1000; Life Technologies) was used as secondary antibody for 1 h in the dark at room temperature. Nuclei were stained using SlowFade® Diamond Antifade Mountant with 4,6-diamidino-2-phenylindole (DAPI) (Life Technologies, Waltham, MA, USA). Images were captured using a confocal laser-scanning microscope (Carl Zeiss LSM 700; Oberkochen, Germany) and ZEN 2012 software.
5
Intracellular Uptake of rM180 in PMA-Differentiated THP-1 Cells
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PMA-differentiated THP-1 cells were seeded onto a glass slide, and stimulated with rM180 for 0, 2, 5, 15, 30 min, 1, 12, and 24 h to observe the intracellular uptake of rM180. After stimulation, cells were fixed using 4% paraformaldehyde (PFA) for 20 min, blocked with Blocking One Histo (Nacalai Tesque) for 5 min at room temperature, treated with 0.5% Triton X-100 (Junsei, Tokyo, Japan), which penetrated into the cells, for 10 min at room temperature, and stained with primary anti-amelogenin (F-11): sc-36528 (1:250; Santa Cruz Biotechnology, Inc, Dallas, TX, USA) antibodies overnight at 4°C. To observe the cell surface expression of MHC II molecules, cells were stained with primary anti-HLA-DR (L243): sc-18875 (1:250; Santa Cruz Biotechnology, Inc.). Subsequently, Alexa Fluor® 594 goat anti-mouse IgG (minimal x-reactivity) (1:1000; BioLegend) was used as secondary antibody for 2 h in the dark at room temperature. The nucleus was stained using SlowFade® Diamond Antifade Mountant with DAPI (Life Technologies, Waltham, MA, USA). The images were analyzed by ZEISS LSM700 (Carl Zeiss, Oberkochen, Germany) and ZEN 2012 software.
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