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Nacalai nonidet p 40
Invasion and adhesion of MKN1 cells and effects of the anti-α3 integrin antibody. A monolayer of mesothelial cells was stimulated with TNF-α (10 ng/mL) for 6 h. ( A ) MKN1 cells (1 × 10 5  cells/0.2 mL) in ASF104 serum-free medium were added to the inner chamber of the reconstituted mesothelium and incubated at 37 °C for 16 h. The cells that had migrated into the outer chamber through the membrane were counted under a microscope. ( B ) Fluorescently labeled MKN1 cells were added to the monolayer of mesothelial cells in a 96-well culture plate, and incubated at 37 °C for 40 min. After non-adherent cells were removed by washing, adherent cells were lysed with 1% Nonidet P-40 and measured with a fluorescence spectrophotometer (Ex = 490 nm, Em = 520 nm). For the inhibition experiments, MKN1 cells were treated with the anti-α3 integrin antibody (SM-T1, 10 μg/mL) at 0 °C for 30 min. Experiments were performed in triplicate, and the data are presented as the mean ± SEM. Statistical data analysis was conducted using the Student’s  t -test. **  p
Nonidet P 40, supplied by Nacalai, used in various techniques. Bioz Stars score: 93/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Stimulation of Peritoneal Mesothelial Cells to Secrete Matrix Metalloproteinase-9 (MMP-9) by TNF-α: A Role in the Invasion of Gastric Carcinoma Cells"

Article Title: Stimulation of Peritoneal Mesothelial Cells to Secrete Matrix Metalloproteinase-9 (MMP-9) by TNF-α: A Role in the Invasion of Gastric Carcinoma Cells

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19123961

Invasion and adhesion of MKN1 cells and effects of the anti-α3 integrin antibody. A monolayer of mesothelial cells was stimulated with TNF-α (10 ng/mL) for 6 h. ( A ) MKN1 cells (1 × 10 5  cells/0.2 mL) in ASF104 serum-free medium were added to the inner chamber of the reconstituted mesothelium and incubated at 37 °C for 16 h. The cells that had migrated into the outer chamber through the membrane were counted under a microscope. ( B ) Fluorescently labeled MKN1 cells were added to the monolayer of mesothelial cells in a 96-well culture plate, and incubated at 37 °C for 40 min. After non-adherent cells were removed by washing, adherent cells were lysed with 1% Nonidet P-40 and measured with a fluorescence spectrophotometer (Ex = 490 nm, Em = 520 nm). For the inhibition experiments, MKN1 cells were treated with the anti-α3 integrin antibody (SM-T1, 10 μg/mL) at 0 °C for 30 min. Experiments were performed in triplicate, and the data are presented as the mean ± SEM. Statistical data analysis was conducted using the Student’s  t -test. **  p
Figure Legend Snippet: Invasion and adhesion of MKN1 cells and effects of the anti-α3 integrin antibody. A monolayer of mesothelial cells was stimulated with TNF-α (10 ng/mL) for 6 h. ( A ) MKN1 cells (1 × 10 5 cells/0.2 mL) in ASF104 serum-free medium were added to the inner chamber of the reconstituted mesothelium and incubated at 37 °C for 16 h. The cells that had migrated into the outer chamber through the membrane were counted under a microscope. ( B ) Fluorescently labeled MKN1 cells were added to the monolayer of mesothelial cells in a 96-well culture plate, and incubated at 37 °C for 40 min. After non-adherent cells were removed by washing, adherent cells were lysed with 1% Nonidet P-40 and measured with a fluorescence spectrophotometer (Ex = 490 nm, Em = 520 nm). For the inhibition experiments, MKN1 cells were treated with the anti-α3 integrin antibody (SM-T1, 10 μg/mL) at 0 °C for 30 min. Experiments were performed in triplicate, and the data are presented as the mean ± SEM. Statistical data analysis was conducted using the Student’s t -test. ** p

Techniques Used: Incubation, Microscopy, Labeling, Fluorescence, Spectrophotometry, Inhibition

2) Product Images from "Stimulation of Peritoneal Mesothelial Cells to Secrete Matrix Metalloproteinase-9 (MMP-9) by TNF-α: A Role in the Invasion of Gastric Carcinoma Cells"

Article Title: Stimulation of Peritoneal Mesothelial Cells to Secrete Matrix Metalloproteinase-9 (MMP-9) by TNF-α: A Role in the Invasion of Gastric Carcinoma Cells

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19123961

Invasion and adhesion of MKN1 cells and effects of the anti-α3 integrin antibody. A monolayer of mesothelial cells was stimulated with TNF-α (10 ng/mL) for 6 h. ( A ) MKN1 cells (1 × 10 5  cells/0.2 mL) in ASF104 serum-free medium were added to the inner chamber of the reconstituted mesothelium and incubated at 37 °C for 16 h. The cells that had migrated into the outer chamber through the membrane were counted under a microscope. ( B ) Fluorescently labeled MKN1 cells were added to the monolayer of mesothelial cells in a 96-well culture plate, and incubated at 37 °C for 40 min. After non-adherent cells were removed by washing, adherent cells were lysed with 1% Nonidet P-40 and measured with a fluorescence spectrophotometer (Ex = 490 nm, Em = 520 nm). For the inhibition experiments, MKN1 cells were treated with the anti-α3 integrin antibody (SM-T1, 10 μg/mL) at 0 °C for 30 min. Experiments were performed in triplicate, and the data are presented as the mean ± SEM. Statistical data analysis was conducted using the Student’s  t -test. **  p
Figure Legend Snippet: Invasion and adhesion of MKN1 cells and effects of the anti-α3 integrin antibody. A monolayer of mesothelial cells was stimulated with TNF-α (10 ng/mL) for 6 h. ( A ) MKN1 cells (1 × 10 5 cells/0.2 mL) in ASF104 serum-free medium were added to the inner chamber of the reconstituted mesothelium and incubated at 37 °C for 16 h. The cells that had migrated into the outer chamber through the membrane were counted under a microscope. ( B ) Fluorescently labeled MKN1 cells were added to the monolayer of mesothelial cells in a 96-well culture plate, and incubated at 37 °C for 40 min. After non-adherent cells were removed by washing, adherent cells were lysed with 1% Nonidet P-40 and measured with a fluorescence spectrophotometer (Ex = 490 nm, Em = 520 nm). For the inhibition experiments, MKN1 cells were treated with the anti-α3 integrin antibody (SM-T1, 10 μg/mL) at 0 °C for 30 min. Experiments were performed in triplicate, and the data are presented as the mean ± SEM. Statistical data analysis was conducted using the Student’s t -test. ** p

Techniques Used: Incubation, Microscopy, Labeling, Fluorescence, Spectrophotometry, Inhibition

3) Product Images from "Constitutive Turnover of Phosphorylation at Thr-412 of Human p57/Coronin-1 Regulates the Interaction with Actin *"

Article Title: Constitutive Turnover of Phosphorylation at Thr-412 of Human p57/Coronin-1 Regulates the Interaction with Actin *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.349829

Transient phosphorylation at Thr-412 of p57/coronin-1 in phagocytosing cells.  Human neutrophils were incubated with opsonized zymosan at 37 °C for 0–30 min and lysed with a buffer containing 1% Nonidet P-40. The lysates were subjected to immunoprecipitation with Dynabeads® protein G/anti-p57/coronin-1 antibody (N7), and the immunoprecipitates were analyzed by SDS-PAGE/immunoblotting with anti-p57/coronin-1 antibody (N7) or anti-phospho-Thr-412 of p57/coronin-1 antibody ( pT412 ). The experiments were repeated three times, and representative results are shown.
Figure Legend Snippet: Transient phosphorylation at Thr-412 of p57/coronin-1 in phagocytosing cells. Human neutrophils were incubated with opsonized zymosan at 37 °C for 0–30 min and lysed with a buffer containing 1% Nonidet P-40. The lysates were subjected to immunoprecipitation with Dynabeads® protein G/anti-p57/coronin-1 antibody (N7), and the immunoprecipitates were analyzed by SDS-PAGE/immunoblotting with anti-p57/coronin-1 antibody (N7) or anti-phospho-Thr-412 of p57/coronin-1 antibody ( pT412 ). The experiments were repeated three times, and representative results are shown.

Techniques Used: Incubation, Immunoprecipitation, SDS Page

4) Product Images from "Endogenous Molecules Stimulating N-Acylethanolamine-Hydrolyzing Acid Amidase (NAAA)"

Article Title: Endogenous Molecules Stimulating N-Acylethanolamine-Hydrolyzing Acid Amidase (NAAA)

Journal: ACS Chemical Neuroscience

doi: 10.1021/cn300007s

Substrate specificity of NAAA in the presenceof different stimulators.Recombinant NAAA (0.3–3 μg of protein) was allowed toreact with the indicated  14 C-labeled NAEs at 100 μMin the presence or absence of 0.1% Nonidet P-40,
Figure Legend Snippet: Substrate specificity of NAAA in the presenceof different stimulators.Recombinant NAAA (0.3–3 μg of protein) was allowed toreact with the indicated 14 C-labeled NAEs at 100 μMin the presence or absence of 0.1% Nonidet P-40,

Techniques Used: Recombinant, Labeling

Effects of various buffers on NAAA activity. Recombinant NAAA (300ng of protein) was allowed to react with 100 μM [ 14 C]PEA in the presence of 3 mM DTT and 0.1% Nonidet P-40 at pH 4.5.The pH was adjusted with the following buffers (100 mM): citrate-Na
Figure Legend Snippet: Effects of various buffers on NAAA activity. Recombinant NAAA (300ng of protein) was allowed to react with 100 μM [ 14 C]PEA in the presence of 3 mM DTT and 0.1% Nonidet P-40 at pH 4.5.The pH was adjusted with the following buffers (100 mM): citrate-Na

Techniques Used: Activity Assay, Recombinant

Effects of various phospholipid classes on NAAA activity.RecombinantNAAA (300 ng of protein) was allowed to react with 100 μM [ 14 C]PEA. (A) The reactions were carried out in the presenceof 3 mM DTT and either Nonidet P-40 at 0.1% (w/v) or the
Figure Legend Snippet: Effects of various phospholipid classes on NAAA activity.RecombinantNAAA (300 ng of protein) was allowed to react with 100 μM [ 14 C]PEA. (A) The reactions were carried out in the presenceof 3 mM DTT and either Nonidet P-40 at 0.1% (w/v) or the

Techniques Used: Activity Assay

Effects of various thiol compounds onNAAA activity. RecombinantNAAA (300 ng of protein) was allowed to react with 100 μM [ 14 C]PEA. (A) The reactions were carried out in the presenceof 0.1% Nonidet P-40 and the indicated compound at 3 mM. (B)
Figure Legend Snippet: Effects of various thiol compounds onNAAA activity. RecombinantNAAA (300 ng of protein) was allowed to react with 100 μM [ 14 C]PEA. (A) The reactions were carried out in the presenceof 0.1% Nonidet P-40 and the indicated compound at 3 mM. (B)

Techniques Used: Activity Assay

5) Product Images from "Stimulation of Peritoneal Mesothelial Cells to Secrete Matrix Metalloproteinase-9 (MMP-9) by TNF-α: A Role in the Invasion of Gastric Carcinoma Cells"

Article Title: Stimulation of Peritoneal Mesothelial Cells to Secrete Matrix Metalloproteinase-9 (MMP-9) by TNF-α: A Role in the Invasion of Gastric Carcinoma Cells

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19123961

Invasion and adhesion of MKN1 cells and effects of the anti-α3 integrin antibody. A monolayer of mesothelial cells was stimulated with TNF-α (10 ng/mL) for 6 h. ( A ) MKN1 cells (1 × 10 5  cells/0.2 mL) in ASF104 serum-free medium were added to the inner chamber of the reconstituted mesothelium and incubated at 37 °C for 16 h. The cells that had migrated into the outer chamber through the membrane were counted under a microscope. ( B ) Fluorescently labeled MKN1 cells were added to the monolayer of mesothelial cells in a 96-well culture plate, and incubated at 37 °C for 40 min. After non-adherent cells were removed by washing, adherent cells were lysed with 1% Nonidet P-40 and measured with a fluorescence spectrophotometer (Ex = 490 nm, Em = 520 nm). For the inhibition experiments, MKN1 cells were treated with the anti-α3 integrin antibody (SM-T1, 10 μg/mL) at 0 °C for 30 min. Experiments were performed in triplicate, and the data are presented as the mean ± SEM. Statistical data analysis was conducted using the Student’s  t -test. **  p
Figure Legend Snippet: Invasion and adhesion of MKN1 cells and effects of the anti-α3 integrin antibody. A monolayer of mesothelial cells was stimulated with TNF-α (10 ng/mL) for 6 h. ( A ) MKN1 cells (1 × 10 5 cells/0.2 mL) in ASF104 serum-free medium were added to the inner chamber of the reconstituted mesothelium and incubated at 37 °C for 16 h. The cells that had migrated into the outer chamber through the membrane were counted under a microscope. ( B ) Fluorescently labeled MKN1 cells were added to the monolayer of mesothelial cells in a 96-well culture plate, and incubated at 37 °C for 40 min. After non-adherent cells were removed by washing, adherent cells were lysed with 1% Nonidet P-40 and measured with a fluorescence spectrophotometer (Ex = 490 nm, Em = 520 nm). For the inhibition experiments, MKN1 cells were treated with the anti-α3 integrin antibody (SM-T1, 10 μg/mL) at 0 °C for 30 min. Experiments were performed in triplicate, and the data are presented as the mean ± SEM. Statistical data analysis was conducted using the Student’s t -test. ** p

Techniques Used: Incubation, Microscopy, Labeling, Fluorescence, Spectrophotometry, Inhibition

6) Product Images from "Stimulation of Peritoneal Mesothelial Cells to Secrete Matrix Metalloproteinase-9 (MMP-9) by TNF-α: A Role in the Invasion of Gastric Carcinoma Cells"

Article Title: Stimulation of Peritoneal Mesothelial Cells to Secrete Matrix Metalloproteinase-9 (MMP-9) by TNF-α: A Role in the Invasion of Gastric Carcinoma Cells

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19123961

Invasion and adhesion of MKN1 cells and effects of the anti-α3 integrin antibody. A monolayer of mesothelial cells was stimulated with TNF-α (10 ng/mL) for 6 h. ( A ) MKN1 cells (1 × 10 5  cells/0.2 mL) in ASF104 serum-free medium were added to the inner chamber of the reconstituted mesothelium and incubated at 37 °C for 16 h. The cells that had migrated into the outer chamber through the membrane were counted under a microscope. ( B ) Fluorescently labeled MKN1 cells were added to the monolayer of mesothelial cells in a 96-well culture plate, and incubated at 37 °C for 40 min. After non-adherent cells were removed by washing, adherent cells were lysed with 1% Nonidet P-40 and measured with a fluorescence spectrophotometer (Ex = 490 nm, Em = 520 nm). For the inhibition experiments, MKN1 cells were treated with the anti-α3 integrin antibody (SM-T1, 10 μg/mL) at 0 °C for 30 min. Experiments were performed in triplicate, and the data are presented as the mean ± SEM. Statistical data analysis was conducted using the Student’s  t -test. **  p
Figure Legend Snippet: Invasion and adhesion of MKN1 cells and effects of the anti-α3 integrin antibody. A monolayer of mesothelial cells was stimulated with TNF-α (10 ng/mL) for 6 h. ( A ) MKN1 cells (1 × 10 5 cells/0.2 mL) in ASF104 serum-free medium were added to the inner chamber of the reconstituted mesothelium and incubated at 37 °C for 16 h. The cells that had migrated into the outer chamber through the membrane were counted under a microscope. ( B ) Fluorescently labeled MKN1 cells were added to the monolayer of mesothelial cells in a 96-well culture plate, and incubated at 37 °C for 40 min. After non-adherent cells were removed by washing, adherent cells were lysed with 1% Nonidet P-40 and measured with a fluorescence spectrophotometer (Ex = 490 nm, Em = 520 nm). For the inhibition experiments, MKN1 cells were treated with the anti-α3 integrin antibody (SM-T1, 10 μg/mL) at 0 °C for 30 min. Experiments were performed in triplicate, and the data are presented as the mean ± SEM. Statistical data analysis was conducted using the Student’s t -test. ** p

Techniques Used: Incubation, Microscopy, Labeling, Fluorescence, Spectrophotometry, Inhibition

Related Articles

Centrifugation:

Article Title: Phospholipase Cϵ Activates Nuclear Factor-κB Signaling by Causing Cytoplasmic Localization of Ribosomal S6 Kinase and Facilitating Its Phosphorylation of Inhibitor κB in Colon Epithelial Cells *
Article Snippet: .. Briefly, Caco2 cells were harvested using a scraper in ice-cold PBS, and, after centrifugation, the cell pellet was resuspended in a lysis buffer (10 m m Hepes, pH 7.9, 10 m m KCl, 1.5 m m MgCl2 , 0.1 m m EDTA, 0.25% (v/v) Nonidet P-40, and protease inhibitor mixture (Nacalai Tesque)). .. The pellet was resuspended in a nuclear extraction buffer (20 m m Hepes, pH 7.9, 420 m m NaCl, 1.5 m m MgCl2 , 0.1 m m EDTA, 1 m m DTT, 25% (v/v) glycerol, and protease inhibitor mixture) and incubated for 30 min, and, after centrifugation at 21,500 × g for 10 min, the supernatant was used as the cytoplasmic fraction.

Chromatography:

Article Title: Endogenous Molecules Stimulating N-Acylethanolamine-Hydrolyzing Acid Amidase (NAAA)
Article Snippet: .. [1-14 C]Palmitic acid was purchasedfrom PerkinElmer Life Science (Boston, MA); [1-14 C]stearicand [1-14 C]arachidonic acids from GE Healthcare (Piscataway,NJ); [1-14 C]oleic acid from Moravek Biochemicals (Brea,CA); nonradiolabeled NAEs from Cayman Chemical (Ann Arbor, MI); bovineserum albumin, 1,2-dioleoyl-PE, 1,2-dioleoyl-PC, 1,2-dipalmitoyl-PG,bovine liver PI, 1,2-dioleoyl-PS, bovine brain SM, 2-mercaptoethanol,dihydrolipoic and α-lipoic acids, and fetal calf serum fromSigma-Aldrich (St. Louis, MO); Triton X-100, Tween 20, DTT, l -cysteine hydrochloride, glutathione, formic and succinic acids,sodium (+)-tartrate, 3(2)- t -butyl-4-hydroxyanisole,ethanolamine, and Dulbecco’s modified Eagle’s mediumfrom Wako Pure Chemical (Osaka, Japan); Nonidet P-40, dl -homocysteine,potassium hydrogen phthalate, and l -(+)-tartaric and 3,3-dimethylglutaricacids from Nacalai Tesque (Kyoto, Japan); n -octyl-β- d -glucoside and CHAPS from Dojindo (Kumamoto, Japan); proteinassay dye reagent concentrate from Bio-Rad (Hercules, CA); precoatedsilica gel 60 F254 aluminum sheets for thin-layer chromatography(20 × 20 cm, 0.2-mm thickness) from Merck (Darmstadt, Germany);Lipofectamine 2000, TRIzol, and pcDNA3.1(+) from Life Technologies(Carlsbad, CA); PrimeScript RT reagent kit and SYBR Premix Ex Taq II from Takara Bio (Ohtsu, Japan); and HEK293 cells fromHealth Science Research Resources Bank (Osaka, Japan). ..

Protease Inhibitor:

Article Title: Phospholipase Cϵ Activates Nuclear Factor-κB Signaling by Causing Cytoplasmic Localization of Ribosomal S6 Kinase and Facilitating Its Phosphorylation of Inhibitor κB in Colon Epithelial Cells *
Article Snippet: .. Briefly, Caco2 cells were harvested using a scraper in ice-cold PBS, and, after centrifugation, the cell pellet was resuspended in a lysis buffer (10 m m Hepes, pH 7.9, 10 m m KCl, 1.5 m m MgCl2 , 0.1 m m EDTA, 0.25% (v/v) Nonidet P-40, and protease inhibitor mixture (Nacalai Tesque)). .. The pellet was resuspended in a nuclear extraction buffer (20 m m Hepes, pH 7.9, 420 m m NaCl, 1.5 m m MgCl2 , 0.1 m m EDTA, 1 m m DTT, 25% (v/v) glycerol, and protease inhibitor mixture) and incubated for 30 min, and, after centrifugation at 21,500 × g for 10 min, the supernatant was used as the cytoplasmic fraction.

Article Title: HPV18 E1^E4 is assembled into aggresome-like compartment and involved in sequestration of viral oncoproteins.
Article Snippet: .. IMMUNOPRECIPITATION AND IMMUNOBLOT Total cell lysates were prepared with triple detergent lysis buffer [150 mM NaCl, 50 mM Tris–HCl (pH 8.0), 0.1% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholate] supplemented with protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan) and 1 mM DTT. .. The cell lysates were centrifuged at 14,000 rpm for 10 min at 4°C, and the supernatants were used for immunoprecipitation and immunoblot.

Immunoprecipitation:

Article Title: HPV18 E1^E4 is assembled into aggresome-like compartment and involved in sequestration of viral oncoproteins.
Article Snippet: .. IMMUNOPRECIPITATION AND IMMUNOBLOT Total cell lysates were prepared with triple detergent lysis buffer [150 mM NaCl, 50 mM Tris–HCl (pH 8.0), 0.1% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholate] supplemented with protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan) and 1 mM DTT. .. The cell lysates were centrifuged at 14,000 rpm for 10 min at 4°C, and the supernatants were used for immunoprecipitation and immunoblot.

other:

Article Title: Stimulation of Peritoneal Mesothelial Cells to Secrete Matrix Metalloproteinase-9 (MMP-9) by TNF-α: A Role in the Invasion of Gastric Carcinoma Cells
Article Snippet: Reagents RPMI1640 medium, trypsin, Triton X-100, Brij 35, TriReagent and hexadimethrine bromide were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Article Title: Stimulation of Peritoneal Mesothelial Cells to Secrete Matrix Metalloproteinase-9 (MMP-9) by TNF-α: A Role in the Invasion of Gastric Carcinoma Cells
Article Snippet: Nonidet P-40 was from Nacalai Tesque (Kyoto, Japan).

Article Title: Stimulation of Peritoneal Mesothelial Cells to Secrete Matrix Metalloproteinase-9 (MMP-9) by TNF-α: A Role in the Invasion of Gastric Carcinoma Cells
Article Snippet: Adherent cells were lysed with 0.1 mL of 1% Nonidet P-40, and the lysates were diluted with 9 volumes of PBS.

Modification:

Article Title: Endogenous Molecules Stimulating N-Acylethanolamine-Hydrolyzing Acid Amidase (NAAA)
Article Snippet: .. [1-14 C]Palmitic acid was purchasedfrom PerkinElmer Life Science (Boston, MA); [1-14 C]stearicand [1-14 C]arachidonic acids from GE Healthcare (Piscataway,NJ); [1-14 C]oleic acid from Moravek Biochemicals (Brea,CA); nonradiolabeled NAEs from Cayman Chemical (Ann Arbor, MI); bovineserum albumin, 1,2-dioleoyl-PE, 1,2-dioleoyl-PC, 1,2-dipalmitoyl-PG,bovine liver PI, 1,2-dioleoyl-PS, bovine brain SM, 2-mercaptoethanol,dihydrolipoic and α-lipoic acids, and fetal calf serum fromSigma-Aldrich (St. Louis, MO); Triton X-100, Tween 20, DTT, l -cysteine hydrochloride, glutathione, formic and succinic acids,sodium (+)-tartrate, 3(2)- t -butyl-4-hydroxyanisole,ethanolamine, and Dulbecco’s modified Eagle’s mediumfrom Wako Pure Chemical (Osaka, Japan); Nonidet P-40, dl -homocysteine,potassium hydrogen phthalate, and l -(+)-tartaric and 3,3-dimethylglutaricacids from Nacalai Tesque (Kyoto, Japan); n -octyl-β- d -glucoside and CHAPS from Dojindo (Kumamoto, Japan); proteinassay dye reagent concentrate from Bio-Rad (Hercules, CA); precoatedsilica gel 60 F254 aluminum sheets for thin-layer chromatography(20 × 20 cm, 0.2-mm thickness) from Merck (Darmstadt, Germany);Lipofectamine 2000, TRIzol, and pcDNA3.1(+) from Life Technologies(Carlsbad, CA); PrimeScript RT reagent kit and SYBR Premix Ex Taq II from Takara Bio (Ohtsu, Japan); and HEK293 cells fromHealth Science Research Resources Bank (Osaka, Japan). ..

Lysis:

Article Title: Phospholipase Cϵ Activates Nuclear Factor-κB Signaling by Causing Cytoplasmic Localization of Ribosomal S6 Kinase and Facilitating Its Phosphorylation of Inhibitor κB in Colon Epithelial Cells *
Article Snippet: .. Briefly, Caco2 cells were harvested using a scraper in ice-cold PBS, and, after centrifugation, the cell pellet was resuspended in a lysis buffer (10 m m Hepes, pH 7.9, 10 m m KCl, 1.5 m m MgCl2 , 0.1 m m EDTA, 0.25% (v/v) Nonidet P-40, and protease inhibitor mixture (Nacalai Tesque)). .. The pellet was resuspended in a nuclear extraction buffer (20 m m Hepes, pH 7.9, 420 m m NaCl, 1.5 m m MgCl2 , 0.1 m m EDTA, 1 m m DTT, 25% (v/v) glycerol, and protease inhibitor mixture) and incubated for 30 min, and, after centrifugation at 21,500 × g for 10 min, the supernatant was used as the cytoplasmic fraction.

Article Title: HPV18 E1^E4 is assembled into aggresome-like compartment and involved in sequestration of viral oncoproteins.
Article Snippet: .. IMMUNOPRECIPITATION AND IMMUNOBLOT Total cell lysates were prepared with triple detergent lysis buffer [150 mM NaCl, 50 mM Tris–HCl (pH 8.0), 0.1% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholate] supplemented with protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan) and 1 mM DTT. .. The cell lysates were centrifuged at 14,000 rpm for 10 min at 4°C, and the supernatants were used for immunoprecipitation and immunoblot.

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    Nacalai nonidet p 40
    Invasion and adhesion of MKN1 cells and effects of the anti-α3 integrin antibody. A monolayer of mesothelial cells was stimulated with TNF-α (10 ng/mL) for 6 h. ( A ) MKN1 cells (1 × 10 5  cells/0.2 mL) in ASF104 serum-free medium were added to the inner chamber of the reconstituted mesothelium and incubated at 37 °C for 16 h. The cells that had migrated into the outer chamber through the membrane were counted under a microscope. ( B ) Fluorescently labeled MKN1 cells were added to the monolayer of mesothelial cells in a 96-well culture plate, and incubated at 37 °C for 40 min. After non-adherent cells were removed by washing, adherent cells were lysed with 1% Nonidet P-40 and measured with a fluorescence spectrophotometer (Ex = 490 nm, Em = 520 nm). For the inhibition experiments, MKN1 cells were treated with the anti-α3 integrin antibody (SM-T1, 10 μg/mL) at 0 °C for 30 min. Experiments were performed in triplicate, and the data are presented as the mean ± SEM. Statistical data analysis was conducted using the Student’s  t -test. **  p
    Nonidet P 40, supplied by Nacalai, used in various techniques. Bioz Stars score: 93/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nonidet p 40/product/Nacalai
    Average 93 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    nonidet p 40 - by Bioz Stars, 2020-11
    93/100 stars
      Buy from Supplier

    93
    Nacalai np 40
    Changes in the viability of F . tularensis SCHU P9 using detergents. (A–C) Bacteria suspended in CDM (A), undiluted FBS (B) and RPMI 1640 containing 10% FBS (C) with and without detergents (1% LDS buffer, 1% <t>NP-40</t> and 1% TritonX-100) were incubated at 4°C for 10 min, 1 h and 1 day. After incubation, the bacteria were immediately centrifuged, and the CFU number of the pellets was calculated. (D–F) Detergents (1% LDS buffer, 1% NP-40 and 1% TritonX-100) were added to bacterial suspensions in CDM (D), undiluted FBS (E) and RPMI 1640 containing 10% FBS (F). The samples were homogenized at 4,200 rpm for 30 s and then immediately cooled on ice. The mean CFU number ± SD of four replicates are shown. Statistical significance was determined by two-way ANOVA (A–C) and one-way ANOVA (D–F) with a post hoc test (*** p
    Np 40, supplied by Nacalai, used in various techniques. Bioz Stars score: 93/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/np 40/product/Nacalai
    Average 93 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    np 40 - by Bioz Stars, 2020-11
    93/100 stars
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    Invasion and adhesion of MKN1 cells and effects of the anti-α3 integrin antibody. A monolayer of mesothelial cells was stimulated with TNF-α (10 ng/mL) for 6 h. ( A ) MKN1 cells (1 × 10 5  cells/0.2 mL) in ASF104 serum-free medium were added to the inner chamber of the reconstituted mesothelium and incubated at 37 °C for 16 h. The cells that had migrated into the outer chamber through the membrane were counted under a microscope. ( B ) Fluorescently labeled MKN1 cells were added to the monolayer of mesothelial cells in a 96-well culture plate, and incubated at 37 °C for 40 min. After non-adherent cells were removed by washing, adherent cells were lysed with 1% Nonidet P-40 and measured with a fluorescence spectrophotometer (Ex = 490 nm, Em = 520 nm). For the inhibition experiments, MKN1 cells were treated with the anti-α3 integrin antibody (SM-T1, 10 μg/mL) at 0 °C for 30 min. Experiments were performed in triplicate, and the data are presented as the mean ± SEM. Statistical data analysis was conducted using the Student’s  t -test. **  p

    Journal: International Journal of Molecular Sciences

    Article Title: Stimulation of Peritoneal Mesothelial Cells to Secrete Matrix Metalloproteinase-9 (MMP-9) by TNF-α: A Role in the Invasion of Gastric Carcinoma Cells

    doi: 10.3390/ijms19123961

    Figure Lengend Snippet: Invasion and adhesion of MKN1 cells and effects of the anti-α3 integrin antibody. A monolayer of mesothelial cells was stimulated with TNF-α (10 ng/mL) for 6 h. ( A ) MKN1 cells (1 × 10 5 cells/0.2 mL) in ASF104 serum-free medium were added to the inner chamber of the reconstituted mesothelium and incubated at 37 °C for 16 h. The cells that had migrated into the outer chamber through the membrane were counted under a microscope. ( B ) Fluorescently labeled MKN1 cells were added to the monolayer of mesothelial cells in a 96-well culture plate, and incubated at 37 °C for 40 min. After non-adherent cells were removed by washing, adherent cells were lysed with 1% Nonidet P-40 and measured with a fluorescence spectrophotometer (Ex = 490 nm, Em = 520 nm). For the inhibition experiments, MKN1 cells were treated with the anti-α3 integrin antibody (SM-T1, 10 μg/mL) at 0 °C for 30 min. Experiments were performed in triplicate, and the data are presented as the mean ± SEM. Statistical data analysis was conducted using the Student’s t -test. ** p

    Article Snippet: Nonidet P-40 was from Nacalai Tesque (Kyoto, Japan).

    Techniques: Incubation, Microscopy, Labeling, Fluorescence, Spectrophotometry, Inhibition

    Invasion and adhesion of MKN1 cells and effects of the anti-α3 integrin antibody. A monolayer of mesothelial cells was stimulated with TNF-α (10 ng/mL) for 6 h. ( A ) MKN1 cells (1 × 10 5  cells/0.2 mL) in ASF104 serum-free medium were added to the inner chamber of the reconstituted mesothelium and incubated at 37 °C for 16 h. The cells that had migrated into the outer chamber through the membrane were counted under a microscope. ( B ) Fluorescently labeled MKN1 cells were added to the monolayer of mesothelial cells in a 96-well culture plate, and incubated at 37 °C for 40 min. After non-adherent cells were removed by washing, adherent cells were lysed with 1% Nonidet P-40 and measured with a fluorescence spectrophotometer (Ex = 490 nm, Em = 520 nm). For the inhibition experiments, MKN1 cells were treated with the anti-α3 integrin antibody (SM-T1, 10 μg/mL) at 0 °C for 30 min. Experiments were performed in triplicate, and the data are presented as the mean ± SEM. Statistical data analysis was conducted using the Student’s  t -test. **  p

    Journal: International Journal of Molecular Sciences

    Article Title: Stimulation of Peritoneal Mesothelial Cells to Secrete Matrix Metalloproteinase-9 (MMP-9) by TNF-α: A Role in the Invasion of Gastric Carcinoma Cells

    doi: 10.3390/ijms19123961

    Figure Lengend Snippet: Invasion and adhesion of MKN1 cells and effects of the anti-α3 integrin antibody. A monolayer of mesothelial cells was stimulated with TNF-α (10 ng/mL) for 6 h. ( A ) MKN1 cells (1 × 10 5 cells/0.2 mL) in ASF104 serum-free medium were added to the inner chamber of the reconstituted mesothelium and incubated at 37 °C for 16 h. The cells that had migrated into the outer chamber through the membrane were counted under a microscope. ( B ) Fluorescently labeled MKN1 cells were added to the monolayer of mesothelial cells in a 96-well culture plate, and incubated at 37 °C for 40 min. After non-adherent cells were removed by washing, adherent cells were lysed with 1% Nonidet P-40 and measured with a fluorescence spectrophotometer (Ex = 490 nm, Em = 520 nm). For the inhibition experiments, MKN1 cells were treated with the anti-α3 integrin antibody (SM-T1, 10 μg/mL) at 0 °C for 30 min. Experiments were performed in triplicate, and the data are presented as the mean ± SEM. Statistical data analysis was conducted using the Student’s t -test. ** p

    Article Snippet: Adherent cells were lysed with 0.1 mL of 1% Nonidet P-40, and the lysates were diluted with 9 volumes of PBS.

    Techniques: Incubation, Microscopy, Labeling, Fluorescence, Spectrophotometry, Inhibition

    Transient phosphorylation at Thr-412 of p57/coronin-1 in phagocytosing cells.  Human neutrophils were incubated with opsonized zymosan at 37 °C for 0–30 min and lysed with a buffer containing 1% Nonidet P-40. The lysates were subjected to immunoprecipitation with Dynabeads® protein G/anti-p57/coronin-1 antibody (N7), and the immunoprecipitates were analyzed by SDS-PAGE/immunoblotting with anti-p57/coronin-1 antibody (N7) or anti-phospho-Thr-412 of p57/coronin-1 antibody ( pT412 ). The experiments were repeated three times, and representative results are shown.

    Journal: The Journal of Biological Chemistry

    Article Title: Constitutive Turnover of Phosphorylation at Thr-412 of Human p57/Coronin-1 Regulates the Interaction with Actin *

    doi: 10.1074/jbc.M112.349829

    Figure Lengend Snippet: Transient phosphorylation at Thr-412 of p57/coronin-1 in phagocytosing cells. Human neutrophils were incubated with opsonized zymosan at 37 °C for 0–30 min and lysed with a buffer containing 1% Nonidet P-40. The lysates were subjected to immunoprecipitation with Dynabeads® protein G/anti-p57/coronin-1 antibody (N7), and the immunoprecipitates were analyzed by SDS-PAGE/immunoblotting with anti-p57/coronin-1 antibody (N7) or anti-phospho-Thr-412 of p57/coronin-1 antibody ( pT412 ). The experiments were repeated three times, and representative results are shown.

    Article Snippet: Nonidet P-40 and Phos-tag® acrylamide were from Nacalai Tesque (Kyoto, Japan) and NARD Institute Ltd. (Amagasaki, Japan), respectively.

    Techniques: Incubation, Immunoprecipitation, SDS Page

    Changes in the viability of F . tularensis SCHU P9 using detergents. (A–C) Bacteria suspended in CDM (A), undiluted FBS (B) and RPMI 1640 containing 10% FBS (C) with and without detergents (1% LDS buffer, 1% NP-40 and 1% TritonX-100) were incubated at 4°C for 10 min, 1 h and 1 day. After incubation, the bacteria were immediately centrifuged, and the CFU number of the pellets was calculated. (D–F) Detergents (1% LDS buffer, 1% NP-40 and 1% TritonX-100) were added to bacterial suspensions in CDM (D), undiluted FBS (E) and RPMI 1640 containing 10% FBS (F). The samples were homogenized at 4,200 rpm for 30 s and then immediately cooled on ice. The mean CFU number ± SD of four replicates are shown. Statistical significance was determined by two-way ANOVA (A–C) and one-way ANOVA (D–F) with a post hoc test (*** p

    Journal: PLoS ONE

    Article Title: Effective methods for the inactivation of Francisella tularensis

    doi: 10.1371/journal.pone.0225177

    Figure Lengend Snippet: Changes in the viability of F . tularensis SCHU P9 using detergents. (A–C) Bacteria suspended in CDM (A), undiluted FBS (B) and RPMI 1640 containing 10% FBS (C) with and without detergents (1% LDS buffer, 1% NP-40 and 1% TritonX-100) were incubated at 4°C for 10 min, 1 h and 1 day. After incubation, the bacteria were immediately centrifuged, and the CFU number of the pellets was calculated. (D–F) Detergents (1% LDS buffer, 1% NP-40 and 1% TritonX-100) were added to bacterial suspensions in CDM (D), undiluted FBS (E) and RPMI 1640 containing 10% FBS (F). The samples were homogenized at 4,200 rpm for 30 s and then immediately cooled on ice. The mean CFU number ± SD of four replicates are shown. Statistical significance was determined by two-way ANOVA (A–C) and one-way ANOVA (D–F) with a post hoc test (*** p

    Article Snippet: Five microliters of F . tularensis SCHU P9 was mixed with 100 μL of 1% Triton X-100, 1% NP-40 and 1% LDS (Nacalai Tesque) buffer supplemented with 1 × sodium dodecyl sulphate buffer, 10% glycerol (Wako) and 0.005% bromophenol blue (63 mM Tris-HCl, pH 6.8; Wako).

    Techniques: Incubation

    The validation of effective treatments for the inactivation in five F . tularensis strains. (A) Bacterial suspensions in CDM were heated at 94°C for 3 min and 56°C for 30 min and immediately cooled on ice. (B) Bacterial suspensions in 1 mL of CDM were filtered through 0.45 and 0.22 μm pore size membrane filters (Merck Millipore). (C) Bacteria spiked into detergents (1% LDS buffer, 1% NP-40 and 1% TritonX-100) were incubated at 4°C for 1 day. (E-F) Bacteria suspended in deionized water (E), CDM (F), and undiluted FBS (G) were aliquoted into four 0.2 mL PCR tubes. These samples were simultaneously radiated with UV light. All treated samples were compared with the control samples. All experiments were performed in four replicates. The mean CFU ± SD are shown.

    Journal: PLoS ONE

    Article Title: Effective methods for the inactivation of Francisella tularensis

    doi: 10.1371/journal.pone.0225177

    Figure Lengend Snippet: The validation of effective treatments for the inactivation in five F . tularensis strains. (A) Bacterial suspensions in CDM were heated at 94°C for 3 min and 56°C for 30 min and immediately cooled on ice. (B) Bacterial suspensions in 1 mL of CDM were filtered through 0.45 and 0.22 μm pore size membrane filters (Merck Millipore). (C) Bacteria spiked into detergents (1% LDS buffer, 1% NP-40 and 1% TritonX-100) were incubated at 4°C for 1 day. (E-F) Bacteria suspended in deionized water (E), CDM (F), and undiluted FBS (G) were aliquoted into four 0.2 mL PCR tubes. These samples were simultaneously radiated with UV light. All treated samples were compared with the control samples. All experiments were performed in four replicates. The mean CFU ± SD are shown.

    Article Snippet: Five microliters of F . tularensis SCHU P9 was mixed with 100 μL of 1% Triton X-100, 1% NP-40 and 1% LDS (Nacalai Tesque) buffer supplemented with 1 × sodium dodecyl sulphate buffer, 10% glycerol (Wako) and 0.005% bromophenol blue (63 mM Tris-HCl, pH 6.8; Wako).

    Techniques: Incubation, Polymerase Chain Reaction