triton x 100 buffer  (Millipore)


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  • 99
    Name:
    Triton X 100
    Description:

    Catalog Number:
    t8532
    Price:
    None
    Applications:
    Detergent used for isoelectric focusing (IEF) and two-dimensional electrophoresis. Concentrations between 1-4% (v/v) are typically used in an IEF gel. Also used for cell permeabilization and protein solubilization including high molecular mass proteins.
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    Structured Review

    Millipore triton x 100 buffer
    Triton X 100

    https://www.bioz.com/result/triton x 100 buffer/product/Millipore
    Average 99 stars, based on 49 article reviews
    Price from $9.99 to $1999.99
    triton x 100 buffer - by Bioz Stars, 2020-07
    99/100 stars

    Images

    1) Product Images from "Coupling of terminal differentiation deficit with neurodegenerative pathology in Vps35-deficient pyramidal neurons"

    Article Title: Coupling of terminal differentiation deficit with neurodegenerative pathology in Vps35-deficient pyramidal neurons

    Journal: Cell Death and Differentiation

    doi: 10.1038/s41418-019-0487-2

    FTD-like neuropathology in  Vps35 Neurod6  neocortex. a  and  e  Western blot analyses of soluble (by 1% Triton X-100) and insoluble fractions (by 7 M Urea, 2 M Thiourea, 4% CHAPS) of homogenates of P14 control and  Vps35 Neurod6  neocortex using indicated antibodies.  b ,  f  Quantification analysis of relative protein expression levels from  a  and  g . ( n  = 4 animals per genotype; two-tailed unpaired  t -test).  c  Tile scan assembly of a coronal section of P14 brain immunostained with anti-P62. Ctx: Cortex; Hip hippocampus and Str Striatum.  d  Representative images of co-immunostaining analysis with antibodies against P62 (green) and p-Tdp43 (purple) of P14 cortical sections.  g  Analysis of  VPS35  mRNA levels in the frontal lobe of cortex (frontal), hippocampus (hippo), and cerebellum from a cohort of eight unaffected (Control), six FTD-TDP patients with Pgrn mutation, and ten FTD-TDP without Pgrn mutation. (Data were from GEO GSE20141). Expression levels are normalized to mean of the unaffected group (One-way ANOVA with Tukey’s multiple comparison test). Scale bars: in  c , 500 μm; in  d , 10 μm. Individual data points were shown as dots with group mean ± s.e.m; * P
    Figure Legend Snippet: FTD-like neuropathology in Vps35 Neurod6 neocortex. a and e Western blot analyses of soluble (by 1% Triton X-100) and insoluble fractions (by 7 M Urea, 2 M Thiourea, 4% CHAPS) of homogenates of P14 control and Vps35 Neurod6 neocortex using indicated antibodies. b , f Quantification analysis of relative protein expression levels from a and g . ( n  = 4 animals per genotype; two-tailed unpaired t -test). c Tile scan assembly of a coronal section of P14 brain immunostained with anti-P62. Ctx: Cortex; Hip hippocampus and Str Striatum. d Representative images of co-immunostaining analysis with antibodies against P62 (green) and p-Tdp43 (purple) of P14 cortical sections. g Analysis of VPS35 mRNA levels in the frontal lobe of cortex (frontal), hippocampus (hippo), and cerebellum from a cohort of eight unaffected (Control), six FTD-TDP patients with Pgrn mutation, and ten FTD-TDP without Pgrn mutation. (Data were from GEO GSE20141). Expression levels are normalized to mean of the unaffected group (One-way ANOVA with Tukey’s multiple comparison test). Scale bars: in c , 500 μm; in d , 10 μm. Individual data points were shown as dots with group mean ± s.e.m; * P

    Techniques Used: Western Blot, Expressing, Two Tailed Test, Immunostaining, Mutagenesis

    2) Product Images from "CD4-Dependent Modulation of HIV-1 Entry by LY6E"

    Article Title: CD4-Dependent Modulation of HIV-1 Entry by LY6E

    Journal: Journal of Virology

    doi: 10.1128/JVI.01866-18

    LY6E is associated with CD4 on the plasma membrane and promotes its internalization. (A) Kinetics of CD4 internalization in the presence or absence of LY6E. CD4 internalization was measured by incubating Jurkat or Jurkat-LY6E cells with a PE-Texas Red-conjugated anti-CD4 antibody (clone S3.5; Invitrogen) on ice for 2 h, followed by switching the temperature to 37°C at different periods of time. At each time point, one half of the harvested cells was treated with pH 3.0 in order to remove the surface CD4, the signals thus reflecting the internalized fraction of CD4. The other half was treated with pH 7.0 to measure the total CD4 level. The CD4 internalized rate was calculated as the ratio of pH 3.0-resistant PE-Texas Red fluorescence intensity versus the total cell-associated fluorescence intensity (pH 7.0). Relative rates of internalization were shown by setting the values of parental Jurkat cells at 60 min without acid wash to 1.0. (B) Effect of different chemical inhibitors on the steady-state level of cell surface CD4 expression. DMSO, 80 μM Dynasore, 10 μM CPZ, or 10 mM MBCD was applied to Jurkat or Jurkat-LY6E cells; 8 h after treatment, cells were collected to determine the cell surface expression of CD4 by flow cytometry. The MFI (geometric mean) of each sample was obtained and compared to the value of DMSO-treated parental cells, which was set to 1.0. (C) Lipid raft flotation assay. Jurkat or Jurkat-LY6E cells were lysed and homogenized in 1% Triton X-100 lysis buffer at 4°C, and harvested lysates were subjected to sucrose cushion ultracentrifugation to determine the water-soluble and detergent-resistant membrane fractions. Samples were collected from the top fraction to the bottom fraction upon completion of ultracentrifugation and subjected to SDS-PAGE; CD4/LY6E expression and distribution was determined by immunoblotting using specific antibodies. (D) Analysis of colocalization between LY6E and CD4. Jurkat-TAg cells (Jurkat cells expressing simian virus 40 [SV40] T antigen but with high transfection efficiency) were cotransfected with plasmids encoding CD4, GFP-GPI, or TIM-1. Thirty-six hours after transfection, cells were fixed and costained with specific antibodies. White arrows indicate colocalized areas of the plasma membrane that were positive for both LY6E and CD4. (E) Randomly selected cells (5 fields) were used for colocalization analysis using NIH ImageJ software, and the Pearson’s correlation coefficients were determined and plotted. (F) Analysis of colocalization between LY6E and CD4 in MDMs. MDMs were permeabilized with a permeabilization buffer (Fermentas) and intracellularly stained with Texas Red-conjugated anti-CD4 and anti-LY6E antibodies, followed by incubating cells with a FITC-conjugated anti-rabbit antibody. The nucleus was stained with DAPI. White arrows indicate colocalized LY6E and CD4. Images were collected under 100× magnification and processed through deconvolution software. *,  P
    Figure Legend Snippet: LY6E is associated with CD4 on the plasma membrane and promotes its internalization. (A) Kinetics of CD4 internalization in the presence or absence of LY6E. CD4 internalization was measured by incubating Jurkat or Jurkat-LY6E cells with a PE-Texas Red-conjugated anti-CD4 antibody (clone S3.5; Invitrogen) on ice for 2 h, followed by switching the temperature to 37°C at different periods of time. At each time point, one half of the harvested cells was treated with pH 3.0 in order to remove the surface CD4, the signals thus reflecting the internalized fraction of CD4. The other half was treated with pH 7.0 to measure the total CD4 level. The CD4 internalized rate was calculated as the ratio of pH 3.0-resistant PE-Texas Red fluorescence intensity versus the total cell-associated fluorescence intensity (pH 7.0). Relative rates of internalization were shown by setting the values of parental Jurkat cells at 60 min without acid wash to 1.0. (B) Effect of different chemical inhibitors on the steady-state level of cell surface CD4 expression. DMSO, 80 μM Dynasore, 10 μM CPZ, or 10 mM MBCD was applied to Jurkat or Jurkat-LY6E cells; 8 h after treatment, cells were collected to determine the cell surface expression of CD4 by flow cytometry. The MFI (geometric mean) of each sample was obtained and compared to the value of DMSO-treated parental cells, which was set to 1.0. (C) Lipid raft flotation assay. Jurkat or Jurkat-LY6E cells were lysed and homogenized in 1% Triton X-100 lysis buffer at 4°C, and harvested lysates were subjected to sucrose cushion ultracentrifugation to determine the water-soluble and detergent-resistant membrane fractions. Samples were collected from the top fraction to the bottom fraction upon completion of ultracentrifugation and subjected to SDS-PAGE; CD4/LY6E expression and distribution was determined by immunoblotting using specific antibodies. (D) Analysis of colocalization between LY6E and CD4. Jurkat-TAg cells (Jurkat cells expressing simian virus 40 [SV40] T antigen but with high transfection efficiency) were cotransfected with plasmids encoding CD4, GFP-GPI, or TIM-1. Thirty-six hours after transfection, cells were fixed and costained with specific antibodies. White arrows indicate colocalized areas of the plasma membrane that were positive for both LY6E and CD4. (E) Randomly selected cells (5 fields) were used for colocalization analysis using NIH ImageJ software, and the Pearson’s correlation coefficients were determined and plotted. (F) Analysis of colocalization between LY6E and CD4 in MDMs. MDMs were permeabilized with a permeabilization buffer (Fermentas) and intracellularly stained with Texas Red-conjugated anti-CD4 and anti-LY6E antibodies, followed by incubating cells with a FITC-conjugated anti-rabbit antibody. The nucleus was stained with DAPI. White arrows indicate colocalized LY6E and CD4. Images were collected under 100× magnification and processed through deconvolution software. *, P

    Techniques Used: Fluorescence, Expressing, Flow Cytometry, Cytometry, Lysis, SDS Page, Transfection, Software, Staining

    3) Product Images from "Acute Blast Injury Reduces Brain Abeta in Two Rodent Species"

    Article Title: Acute Blast Injury Reduces Brain Abeta in Two Rodent Species

    Journal: Frontiers in Neurology

    doi: 10.3389/fneur.2012.00177

    Enzyme-linked immunosorbent assays for Aβ 40 (A,B) and 42 (C,D) were performed on Triton X-100 brain extracts from rats that were subjected to blast exposures of 36.6, 74.5, or 116.7 kPa and harvested at 24 hours (24 h) or 1 week (1 wk) post-blast exposure . Controls consisted of sham exposed rats harvested at 24 h ( n  = 4) or 1 week ( n  = 5) post-exposure. Because neither Aβ 40 ( p  = 0.29, unpaired  t -test) nor 42 ( p  = 0.84) levels differed between the controls harvested at 24 h and 1 week, the two groups were pooled and treated as a single control group ( n  = 9). Numbers of samples in each group are indicated in Table   1 . Values are presented ± the SEM. Asterisk indicates values that were significantly different from controls based on Dunnett’s test. Note the decrease in Aβ 42 in rats exposed to a 36.6 kPa blast at 24 h and 1 week post-exposure as well as 74.5 kPa exposed rats at 24 h. By contrast Aβ 40 was decreased in only the 74.5 kPa exposed rats at 1 week. Further statistical comparisons are discussed in the text and presented in Table   1 .
    Figure Legend Snippet: Enzyme-linked immunosorbent assays for Aβ 40 (A,B) and 42 (C,D) were performed on Triton X-100 brain extracts from rats that were subjected to blast exposures of 36.6, 74.5, or 116.7 kPa and harvested at 24 hours (24 h) or 1 week (1 wk) post-blast exposure . Controls consisted of sham exposed rats harvested at 24 h ( n  = 4) or 1 week ( n  = 5) post-exposure. Because neither Aβ 40 ( p  = 0.29, unpaired t -test) nor 42 ( p  = 0.84) levels differed between the controls harvested at 24 h and 1 week, the two groups were pooled and treated as a single control group ( n  = 9). Numbers of samples in each group are indicated in Table 1 . Values are presented ± the SEM. Asterisk indicates values that were significantly different from controls based on Dunnett’s test. Note the decrease in Aβ 42 in rats exposed to a 36.6 kPa blast at 24 h and 1 week post-exposure as well as 74.5 kPa exposed rats at 24 h. By contrast Aβ 40 was decreased in only the 74.5 kPa exposed rats at 1 week. Further statistical comparisons are discussed in the text and presented in Table 1 .

    Techniques Used:

    Results of Aβ 40 (A–E) and 42 (F–J) ELISAs on Triton X-100 brain extracts are presented for rats exposed to frontal or side blast exposures of 36.6 (A,F), 74.5 (B,C,G,H), or 116.7 kPa (D,E,I,J) harvested at 24 hours (24 h) or 1 week (1 wk) post-blast exposure . Values are presented ± the SEM. There were no statistically significant differences between any of the pair wise comparisons (unpaired  t -tests) demonstrating that orientation to the blast had no effect on levels of Aβ 40 or 42. Further statistical tests are discussed in the text. Sample sizes were:  (A,F)  (6 frontal and 4 side),  (B,G)  (8 frontal and 10 side),  (C,H)  (3 frontal and 5 side),  (D,I)  (5 frontal and 4 side), and  (E,J)  (4 frontal and 5 side).
    Figure Legend Snippet: Results of Aβ 40 (A–E) and 42 (F–J) ELISAs on Triton X-100 brain extracts are presented for rats exposed to frontal or side blast exposures of 36.6 (A,F), 74.5 (B,C,G,H), or 116.7 kPa (D,E,I,J) harvested at 24 hours (24 h) or 1 week (1 wk) post-blast exposure . Values are presented ± the SEM. There were no statistically significant differences between any of the pair wise comparisons (unpaired t -tests) demonstrating that orientation to the blast had no effect on levels of Aβ 40 or 42. Further statistical tests are discussed in the text. Sample sizes were: (A,F) (6 frontal and 4 side), (B,G) (8 frontal and 10 side), (C,H) (3 frontal and 5 side), (D,I) (5 frontal and 4 side), and (E,J) (4 frontal and 5 side).

    Techniques Used:

    Western blotting was performed on hemi brain Triton X-100 extracts from sham exposed (controls) or rats exposed to 36.6 kPa blast injury harvested at 24 h (A) or 1 week post-blast exposure (B) . The top panel in each set shows blotting with an antibody that recognizes APP. In the lower panels the blots were reprobed for β-tubulin (β-tub) as a loading control. Levels of APP are expressed as the ratio of APP to β-tubulin (±SEM) for the experiments in  (A)  in  (C,D)  and for  (B)  in  (E,F) . Asterisk indicates  p  = 0.05 or less vs. control (unpaired  t -test). Note the increase in APP levels at both 24 h and 1 week post-blast exposure.
    Figure Legend Snippet: Western blotting was performed on hemi brain Triton X-100 extracts from sham exposed (controls) or rats exposed to 36.6 kPa blast injury harvested at 24 h (A) or 1 week post-blast exposure (B) . The top panel in each set shows blotting with an antibody that recognizes APP. In the lower panels the blots were reprobed for β-tubulin (β-tub) as a loading control. Levels of APP are expressed as the ratio of APP to β-tubulin (±SEM) for the experiments in (A) in (C,D) and for (B) in (E,F) . Asterisk indicates p  = 0.05 or less vs. control (unpaired t -test). Note the increase in APP levels at both 24 h and 1 week post-blast exposure.

    Techniques Used: Western Blot

    Western blotting was performed on hemi brain Triton X-100 extracts from sham exposed (controls) or rats exposed to 36.6 kPa blast injury harvested at 24 h (A) or 1 week post-blast exposure (B) . The top panel in each set shows blotting with an antibody that recognizes BACE1. In the lower panels the blots were reprobed for β-tubulin (β-tub) as a loading control. Levels of BACE1 are expressed as the ratio of BACE1 to β-tubulin (±SEM) for the experiments in  (A)  in  (C,D)  and for  (B)  in  (E,F) . Note the lack of change in BACE1 levels in rats exposed to a 36.6 kPa blast at either 24 h or 1 week.
    Figure Legend Snippet: Western blotting was performed on hemi brain Triton X-100 extracts from sham exposed (controls) or rats exposed to 36.6 kPa blast injury harvested at 24 h (A) or 1 week post-blast exposure (B) . The top panel in each set shows blotting with an antibody that recognizes BACE1. In the lower panels the blots were reprobed for β-tubulin (β-tub) as a loading control. Levels of BACE1 are expressed as the ratio of BACE1 to β-tubulin (±SEM) for the experiments in (A) in (C,D) and for (B) in (E,F) . Note the lack of change in BACE1 levels in rats exposed to a 36.6 kPa blast at either 24 h or 1 week.

    Techniques Used: Western Blot

    4) Product Images from "Limited forward trafficking of connexin 43 reduces cell-cell coupling in stressed human and mouse myocardium"

    Article Title: Limited forward trafficking of connexin 43 reduces cell-cell coupling in stressed human and mouse myocardium

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI39740

    Levels of Cx43 and EB1 at the intercalated disc are reduced with end-stage ischemic cardiomyopathy. Immunofluorescence labeling of cryosections from snap-frozen non-failing and end-stage ischemic explanted human heart tissue. ( A ) Human heart Cx43 immunofluorescence. N-cadherin in red and Cx43 in green, with enlarged overlay images. N-cadherin was used as a marker for the intercalated disc (arrows). ( B ) Cx43 at intercalated discs. Quantification of Cx43 fluorescence intensity in regions also positive for N-cadherin expression. ( C ) Tissue fractionation. Triton X-100–based fractionation of soluble (cytoplasmic) and insoluble (junctional) protein from tissue detected by Western blot analysis, quantified in bar charts. ( D ) EB1 immunofluorescence. N-cadherin in red and EB1 in green with enlarged panels at right displaying comparable intercalated discs. ( E ) EB1 enrichment at intercalated discs. Quantification of EB1 fluorescence intensity in regions also positive for N-cadherin expression. Original magnification, ×60. Scale bars: 10 μm. Data are representative of 4 non-failing and 4 end-stage ischemic explanted hearts. Statistical analysis was performed using the Student’s unpaired  t  test. Values represent mean ± SEM. * P
    Figure Legend Snippet: Levels of Cx43 and EB1 at the intercalated disc are reduced with end-stage ischemic cardiomyopathy. Immunofluorescence labeling of cryosections from snap-frozen non-failing and end-stage ischemic explanted human heart tissue. ( A ) Human heart Cx43 immunofluorescence. N-cadherin in red and Cx43 in green, with enlarged overlay images. N-cadherin was used as a marker for the intercalated disc (arrows). ( B ) Cx43 at intercalated discs. Quantification of Cx43 fluorescence intensity in regions also positive for N-cadherin expression. ( C ) Tissue fractionation. Triton X-100–based fractionation of soluble (cytoplasmic) and insoluble (junctional) protein from tissue detected by Western blot analysis, quantified in bar charts. ( D ) EB1 immunofluorescence. N-cadherin in red and EB1 in green with enlarged panels at right displaying comparable intercalated discs. ( E ) EB1 enrichment at intercalated discs. Quantification of EB1 fluorescence intensity in regions also positive for N-cadherin expression. Original magnification, ×60. Scale bars: 10 μm. Data are representative of 4 non-failing and 4 end-stage ischemic explanted hearts. Statistical analysis was performed using the Student’s unpaired t test. Values represent mean ± SEM. * P

    Techniques Used: Immunofluorescence, Labeling, Marker, Fluorescence, Expressing, Fractionation, Western Blot

    5) Product Images from "Amyloid-?1–42 Slows Clearance of Synaptically Released Glutamate by Mislocalizing Astrocytic GLT-1"

    Article Title: Amyloid-?1–42 Slows Clearance of Synaptically Released Glutamate by Mislocalizing Astrocytic GLT-1

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.5274-12.2013

    Pretreating hippocampal slices with Trolox rescues Aβ 1–42 -induced GLT-1 Triton X-100 detergent-insolubility and ubiquitination.  A , Representative Western blots show GLT-1 levels in protein lysates first subjected to three serial 1% Triton X-100 extractions to remove Triton X-100-soluble proteins. Aβ 1–42  increased detergent-insoluble GLT-1 levels compared with control slices. Trolox reduced GLT-1 detergent-insolubility to control levels. Prestaining with Sypro confirmed equivalent protein loading among lanes.  B , Aβ 1–42  increased Triton X-100-insoluble GLT-1 (left) and was rescued by Trolox pretreatment (Ctrl, Aβ 1–42 , and Aβ 1–42  pretreated 1 h before and during 30 min Aβ 1–42  treatment;  n  = 9, 50, and 18, respectively;  F (2,74)  = 13.605,  p
    Figure Legend Snippet: Pretreating hippocampal slices with Trolox rescues Aβ 1–42 -induced GLT-1 Triton X-100 detergent-insolubility and ubiquitination. A , Representative Western blots show GLT-1 levels in protein lysates first subjected to three serial 1% Triton X-100 extractions to remove Triton X-100-soluble proteins. Aβ 1–42 increased detergent-insoluble GLT-1 levels compared with control slices. Trolox reduced GLT-1 detergent-insolubility to control levels. Prestaining with Sypro confirmed equivalent protein loading among lanes. B , Aβ 1–42 increased Triton X-100-insoluble GLT-1 (left) and was rescued by Trolox pretreatment (Ctrl, Aβ 1–42 , and Aβ 1–42 pretreated 1 h before and during 30 min Aβ 1–42 treatment; n = 9, 50, and 18, respectively; F (2,74) = 13.605, p

    Techniques Used: Western Blot

    6) Product Images from "Interferon-γ Induces Immunoproteasomes and the Presentation of MHC I-Associated Peptides on Human Salivary Gland Cells"

    Article Title: Interferon-γ Induces Immunoproteasomes and the Presentation of MHC I-Associated Peptides on Human Salivary Gland Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0102878

    IFN-γ exposure increases immunoproteasome activities in HSG cells. The cells were treated with IFN-γ (250 U/ml) for 24–96 hrs, lysed with triton X-100, and then subjected to proteasome activity assay using fluorogenic substrate, Suc-LLVY-AMC. At each experimental condition, three cell lysates samples obtained from three separate T25 flasks (n = 3) were analyzed. **, p
    Figure Legend Snippet: IFN-γ exposure increases immunoproteasome activities in HSG cells. The cells were treated with IFN-γ (250 U/ml) for 24–96 hrs, lysed with triton X-100, and then subjected to proteasome activity assay using fluorogenic substrate, Suc-LLVY-AMC. At each experimental condition, three cell lysates samples obtained from three separate T25 flasks (n = 3) were analyzed. **, p

    Techniques Used: Activity Assay

    7) Product Images from "Palmitoylation of A-Kinase Anchoring Protein 79/150 Regulates Dendritic Endosomal Targeting and Synaptic Plasticity Mechanisms"

    Article Title: Palmitoylation of A-Kinase Anchoring Protein 79/150 Regulates Dendritic Endosomal Targeting and Synaptic Plasticity Mechanisms

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.0784-12.2012

    AKAP79 palmitoylation confers resistance to Triton X-100 extraction and opposes removal from dendritic spines with NMDA-cLTD stimulation
    Figure Legend Snippet: AKAP79 palmitoylation confers resistance to Triton X-100 extraction and opposes removal from dendritic spines with NMDA-cLTD stimulation

    Techniques Used:

    Related Articles

    MTT Assay:

    Article Title: Antiviral Activity of a Turbot (Scophthalmus maximus) NK-Lysin Peptide by Inhibition of Low-pH Virus-Induced Membrane Fusion
    Article Snippet: .. Carboxyfluorescein, 1,6-Diphenyl-1,3,5-hexatriene (DPH), thiazolyl blue tetrazolium bromide (MTT), formalin, triton X-100 and chloroform were obtained from Sigma-Aldrich (Sant Louise, MO, USA). .. Anhydrous dimethyl sulfoxide (DMSO) was purchased from Merck (Kenilworth, NJ, USA).

    Immunocytochemistry:

    Article Title: Synthesis of the Ca2+-mobilizing messengers NAADP and cADPR by intracellular CD38 enzyme in the mouse heart: Role in β-adrenoceptor signaling
    Article Snippet: .. Immunocytochemistry studies Cells were fixed in in 4% paraformaldehyde/PBS for 15 min, washed in PBS (three changes, 5 min each), permeabilized using 0.1% Triton X-100 (Sigma-Aldrich) for 10 min, washed in PBS, and blocked with 1% bovine serum albumin for 60 min before being incubated with the primary antibody at 4 °C overnight. .. The next day, cells were first washed with PBS before being incubated with secondary antibody at room temperature for 2 h (either Alexa Fluor 488–conjugated donkey anti-rabbit or Dylight 550 donkey anti-mouse, 1:1000 dilution) and then washed.

    Concentration Assay:

    Article Title: KAR5 Encodes a Novel Pheromone-inducible Protein Required for Homotypic Nuclear Fusion
    Article Snippet: .. Bacterial pellets were resuspended in 1/50 culture volume of 16 mM Na2 HPO4 , 4 mM NaH2 PO4 , 150 mM NaCl, 1% Triton X-100 containing 2 μg/ml final concentration each of chymostatin, leupeptin, aprotinin, pepstatin A ( Sigma Chemical Co. , St. Louis, MO), and AEBSF ( Calbiochem Corp. , La Jolla, CA). .. After centrifugation at 12,000 g at 4°C, the pellet was resuspended in Laemmli sample buffer ( ) and boiled for 15 min. GST–Kar5p was resolved on a 10% SDS-PAGE and transferred to nitrocellulose (Schleicher & Schuell, Inc., Keene, NH), and the band corresponding to GST–Kar5p was excised.

    Article Title: Characterization of RNA in Saliva
    Article Snippet: .. incubation of saliva with triton x-100 We added Triton X-100 (Sigma) to saliva to a final concentration of 10 mL/L and incubated the samples at room temperature for up to 20 min. .. When RNA was isolated from these samples, we added water and Triton X-100 to Triton-added and water-added samples, respectively, to balance the chemical compositions between samples.

    Blocking Assay:

    Article Title: Bioengineering the microanatomy of human skin
    Article Snippet: .. Samples were then incubated and permeabilised for 1 h in blocking buffer: 20% neonatal calf serum (NCS, Sigma‐Aldrich) in 0.4% Triton X‐100 (Sigma‐Aldrich) in phosphate‐buffered saline (PBS). .. Primary antibodies diluted in blocking buffer (Table ) were incubated with the samples overnight at 4 °C, followed by three washes in PBS.

    Purification:

    Article Title: Resistive-Pulse Analysis of Single Phospholipid Vesicles Using Quartz Nanochannels
    Article Snippet: .. KC1 (JT Baker), triton X-100 (Sigma-Aldrich), 10 × PBS OmniPur liquid concentrate (EMD Chemicals), 1-α-phosphatidylcholine (PC), and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (Avanti Polar Lipids) were all reagent grade and used without further purification. .. PS microspheres with nominal sizes of 175 and 356 nm were purchased from Poly-sciences Inc. Quartz capillary tubing (o.d.

    Immunoprecipitation:

    Article Title: AMPA Receptor Synaptic Targeting Regulated by Stargazin Interactions with the Golgi-Resident PDZ Protein nPIST
    Article Snippet: .. For immunoprecipitation experiments, the samples were lysed in TEE containing 1% Triton X-100, 10 μg/ml aprotinin (Sigma), 10 μg/ml leupeptin (Sigma), and 1 m m PMSF (Sigma), then incubated with 2 μg of the appropriate antibodies for 1 hr at 4°C. .. After the addition of 20 μl of protein A-Sepharose beads (Sigma), samples were incubated for 1 hr at 4°C.

    Incubation:

    Article Title: AMPA Receptor Synaptic Targeting Regulated by Stargazin Interactions with the Golgi-Resident PDZ Protein nPIST
    Article Snippet: .. For immunoprecipitation experiments, the samples were lysed in TEE containing 1% Triton X-100, 10 μg/ml aprotinin (Sigma), 10 μg/ml leupeptin (Sigma), and 1 m m PMSF (Sigma), then incubated with 2 μg of the appropriate antibodies for 1 hr at 4°C. .. After the addition of 20 μl of protein A-Sepharose beads (Sigma), samples were incubated for 1 hr at 4°C.

    Article Title: Bioengineering the microanatomy of human skin
    Article Snippet: .. Samples were then incubated and permeabilised for 1 h in blocking buffer: 20% neonatal calf serum (NCS, Sigma‐Aldrich) in 0.4% Triton X‐100 (Sigma‐Aldrich) in phosphate‐buffered saline (PBS). .. Primary antibodies diluted in blocking buffer (Table ) were incubated with the samples overnight at 4 °C, followed by three washes in PBS.

    Article Title: Synthesis of the Ca2+-mobilizing messengers NAADP and cADPR by intracellular CD38 enzyme in the mouse heart: Role in β-adrenoceptor signaling
    Article Snippet: .. Immunocytochemistry studies Cells were fixed in in 4% paraformaldehyde/PBS for 15 min, washed in PBS (three changes, 5 min each), permeabilized using 0.1% Triton X-100 (Sigma-Aldrich) for 10 min, washed in PBS, and blocked with 1% bovine serum albumin for 60 min before being incubated with the primary antibody at 4 °C overnight. .. The next day, cells were first washed with PBS before being incubated with secondary antibody at room temperature for 2 h (either Alexa Fluor 488–conjugated donkey anti-rabbit or Dylight 550 donkey anti-mouse, 1:1000 dilution) and then washed.

    Article Title: Characterization of RNA in Saliva
    Article Snippet: .. incubation of saliva with triton x-100 We added Triton X-100 (Sigma) to saliva to a final concentration of 10 mL/L and incubated the samples at room temperature for up to 20 min. .. When RNA was isolated from these samples, we added water and Triton X-100 to Triton-added and water-added samples, respectively, to balance the chemical compositions between samples.

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    Millipore triton x 100
    α7nAChR is associated with detergent-resistant membrane lipid microdomains. A membrane fraction from St 40–42 CGs was prepared by gradient centrifugation, incubated with Triton X-100 on ice for 30 min, and subjected to discontinuous gradient centrifugation (35–30-0% Optiprep) at 285,000 ×  g  for 4 hr at 4°C. Six fractions were collected from the top ( fraction 1 ) to the bottom ( fraction 6 ). α7nAChR was isolated from each sample with αBTX-conjugated Actigel, separated on 10% SDS-PAGE, and analyzed by Western blotting with a monoclonal antibody against the α7nAChR. An aliquot of each fraction was also analyzed by Western blotting with a polyclonal antibody against caveolin. The Western blots indicate that the bulk of α7nAChR floats to the lower density fractions ( fraction 1, 2 ), indicating its association with detergent-resistant membrane structures. The accumulation of the integral lipid raft protein caveolin in the first two fractions confirms the migration of the detergent-insoluble components of the membrane to the low-density portion of the gradient.
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    Millipore immunoprecipitation buffer
    Binding of Wt1 protein to the 5′-flanking region of the Adamts16 gene. ChIP was performed to detect Wt1 protein bound to the 5′-flanking region of the Adamts16 gene in its native chromosomal configuration. The drawing ( A ) delineates the three predicted Wt1 binding sites ( Wt1-A , Wt1-B , and Wt1-C ) in the promoter and 5′-UTR of the Adamts16 gene and allocates the PCR primers used for DNA amplification. Specific antibodies against Wt1 and histone proteins were chosen for <t>immunoprecipitation</t> of M15 whole cell lysates. Amplicons encompassing the 5′-flanking region of the Adamts16 gene were enriched ∼2.5-fold with the use of anti-Wt1 antibody compared with normal rabbit IgG (NRb-IgG). No differences in actin DNA were observed between anti-Wt1 antibody and normal rabbit IgG ( B ). The gene encoding anti-Müllerian hormone receptor 2 ( Amhr2 ), served as a positive control ( B ). Binding of Wt1(−KTS) protein to the Adamts16 promoter was confirmed in stimulated UB27 cells ( C ). Wt1(+KTS) protein failed to interact with the promoter of the Adamts16 gene in UD28 cells ( D ). Data shown are means ± S.E. ( error bars ). Statistical significances are indicated by asterisks (*, p
    Immunoprecipitation Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    FTD-like neuropathology in  Vps35 Neurod6  neocortex. a  and  e  Western blot analyses of soluble (by 1% Triton X-100) and insoluble fractions (by 7 M Urea, 2 M Thiourea, 4% CHAPS) of homogenates of P14 control and  Vps35 Neurod6  neocortex using indicated antibodies.  b ,  f  Quantification analysis of relative protein expression levels from  a  and  g . ( n  = 4 animals per genotype; two-tailed unpaired  t -test).  c  Tile scan assembly of a coronal section of P14 brain immunostained with anti-P62. Ctx: Cortex; Hip hippocampus and Str Striatum.  d  Representative images of co-immunostaining analysis with antibodies against P62 (green) and p-Tdp43 (purple) of P14 cortical sections.  g  Analysis of  VPS35  mRNA levels in the frontal lobe of cortex (frontal), hippocampus (hippo), and cerebellum from a cohort of eight unaffected (Control), six FTD-TDP patients with Pgrn mutation, and ten FTD-TDP without Pgrn mutation. (Data were from GEO GSE20141). Expression levels are normalized to mean of the unaffected group (One-way ANOVA with Tukey’s multiple comparison test). Scale bars: in  c , 500 μm; in  d , 10 μm. Individual data points were shown as dots with group mean ± s.e.m; * P
    Triton X 100 Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    α7nAChR is associated with detergent-resistant membrane lipid microdomains. A membrane fraction from St 40–42 CGs was prepared by gradient centrifugation, incubated with Triton X-100 on ice for 30 min, and subjected to discontinuous gradient centrifugation (35–30-0% Optiprep) at 285,000 ×  g  for 4 hr at 4°C. Six fractions were collected from the top ( fraction 1 ) to the bottom ( fraction 6 ). α7nAChR was isolated from each sample with αBTX-conjugated Actigel, separated on 10% SDS-PAGE, and analyzed by Western blotting with a monoclonal antibody against the α7nAChR. An aliquot of each fraction was also analyzed by Western blotting with a polyclonal antibody against caveolin. The Western blots indicate that the bulk of α7nAChR floats to the lower density fractions ( fraction 1, 2 ), indicating its association with detergent-resistant membrane structures. The accumulation of the integral lipid raft protein caveolin in the first two fractions confirms the migration of the detergent-insoluble components of the membrane to the low-density portion of the gradient.

    Journal: The Journal of Neuroscience

    Article Title: Membrane Lipid Rafts Are Necessary for the Maintenance of the α7 Nicotinic Acetylcholine Receptor in Somatic Spines of Ciliary Neurons

    doi: 10.1523/JNEUROSCI.21-02-00504.2001

    Figure Lengend Snippet: α7nAChR is associated with detergent-resistant membrane lipid microdomains. A membrane fraction from St 40–42 CGs was prepared by gradient centrifugation, incubated with Triton X-100 on ice for 30 min, and subjected to discontinuous gradient centrifugation (35–30-0% Optiprep) at 285,000 × g for 4 hr at 4°C. Six fractions were collected from the top ( fraction 1 ) to the bottom ( fraction 6 ). α7nAChR was isolated from each sample with αBTX-conjugated Actigel, separated on 10% SDS-PAGE, and analyzed by Western blotting with a monoclonal antibody against the α7nAChR. An aliquot of each fraction was also analyzed by Western blotting with a polyclonal antibody against caveolin. The Western blots indicate that the bulk of α7nAChR floats to the lower density fractions ( fraction 1, 2 ), indicating its association with detergent-resistant membrane structures. The accumulation of the integral lipid raft protein caveolin in the first two fractions confirms the migration of the detergent-insoluble components of the membrane to the low-density portion of the gradient.

    Article Snippet: Thereafter, the sample was adjusted to 35% Optiprep, overlayered with 2.5 ml of 30% Optiprep plus 0.1% Triton X-100 and 0.5 ml of homogenization buffer plus 0.1% Triton X-100, and spun as described above for 4 hr.

    Techniques: Gradient Centrifugation, Incubation, Isolation, SDS Page, Western Blot, Migration

    Binding of Wt1 protein to the 5′-flanking region of the Adamts16 gene. ChIP was performed to detect Wt1 protein bound to the 5′-flanking region of the Adamts16 gene in its native chromosomal configuration. The drawing ( A ) delineates the three predicted Wt1 binding sites ( Wt1-A , Wt1-B , and Wt1-C ) in the promoter and 5′-UTR of the Adamts16 gene and allocates the PCR primers used for DNA amplification. Specific antibodies against Wt1 and histone proteins were chosen for immunoprecipitation of M15 whole cell lysates. Amplicons encompassing the 5′-flanking region of the Adamts16 gene were enriched ∼2.5-fold with the use of anti-Wt1 antibody compared with normal rabbit IgG (NRb-IgG). No differences in actin DNA were observed between anti-Wt1 antibody and normal rabbit IgG ( B ). The gene encoding anti-Müllerian hormone receptor 2 ( Amhr2 ), served as a positive control ( B ). Binding of Wt1(−KTS) protein to the Adamts16 promoter was confirmed in stimulated UB27 cells ( C ). Wt1(+KTS) protein failed to interact with the promoter of the Adamts16 gene in UD28 cells ( D ). Data shown are means ± S.E. ( error bars ). Statistical significances are indicated by asterisks (*, p

    Journal: The Journal of Biological Chemistry

    Article Title: Transcriptional Regulation by the Wilms Tumor Protein, Wt1, Suggests a Role of the Metalloproteinase Adamts16 in Murine Genitourinary Development *

    doi: 10.1074/jbc.M113.464644

    Figure Lengend Snippet: Binding of Wt1 protein to the 5′-flanking region of the Adamts16 gene. ChIP was performed to detect Wt1 protein bound to the 5′-flanking region of the Adamts16 gene in its native chromosomal configuration. The drawing ( A ) delineates the three predicted Wt1 binding sites ( Wt1-A , Wt1-B , and Wt1-C ) in the promoter and 5′-UTR of the Adamts16 gene and allocates the PCR primers used for DNA amplification. Specific antibodies against Wt1 and histone proteins were chosen for immunoprecipitation of M15 whole cell lysates. Amplicons encompassing the 5′-flanking region of the Adamts16 gene were enriched ∼2.5-fold with the use of anti-Wt1 antibody compared with normal rabbit IgG (NRb-IgG). No differences in actin DNA were observed between anti-Wt1 antibody and normal rabbit IgG ( B ). The gene encoding anti-Müllerian hormone receptor 2 ( Amhr2 ), served as a positive control ( B ). Binding of Wt1(−KTS) protein to the Adamts16 promoter was confirmed in stimulated UB27 cells ( C ). Wt1(+KTS) protein failed to interact with the promoter of the Adamts16 gene in UD28 cells ( D ). Data shown are means ± S.E. ( error bars ). Statistical significances are indicated by asterisks (*, p

    Article Snippet: The supernatants were diluted in immunoprecipitation buffer (0.01% SDS, 1.1% Triton X-100, 1.2 m m EDTA, 16.7 m m Tris-HCl, pH 8.1) and precleared for 1 h at 4 °C with DNA-blocked protein G-agarose (Millipore).

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification, Immunoprecipitation, Positive Control

    FTD-like neuropathology in  Vps35 Neurod6  neocortex. a  and  e  Western blot analyses of soluble (by 1% Triton X-100) and insoluble fractions (by 7 M Urea, 2 M Thiourea, 4% CHAPS) of homogenates of P14 control and  Vps35 Neurod6  neocortex using indicated antibodies.  b ,  f  Quantification analysis of relative protein expression levels from  a  and  g . ( n  = 4 animals per genotype; two-tailed unpaired  t -test).  c  Tile scan assembly of a coronal section of P14 brain immunostained with anti-P62. Ctx: Cortex; Hip hippocampus and Str Striatum.  d  Representative images of co-immunostaining analysis with antibodies against P62 (green) and p-Tdp43 (purple) of P14 cortical sections.  g  Analysis of  VPS35  mRNA levels in the frontal lobe of cortex (frontal), hippocampus (hippo), and cerebellum from a cohort of eight unaffected (Control), six FTD-TDP patients with Pgrn mutation, and ten FTD-TDP without Pgrn mutation. (Data were from GEO GSE20141). Expression levels are normalized to mean of the unaffected group (One-way ANOVA with Tukey’s multiple comparison test). Scale bars: in  c , 500 μm; in  d , 10 μm. Individual data points were shown as dots with group mean ± s.e.m; * P

    Journal: Cell Death and Differentiation

    Article Title: Coupling of terminal differentiation deficit with neurodegenerative pathology in Vps35-deficient pyramidal neurons

    doi: 10.1038/s41418-019-0487-2

    Figure Lengend Snippet: FTD-like neuropathology in Vps35 Neurod6 neocortex. a and e Western blot analyses of soluble (by 1% Triton X-100) and insoluble fractions (by 7 M Urea, 2 M Thiourea, 4% CHAPS) of homogenates of P14 control and Vps35 Neurod6 neocortex using indicated antibodies. b , f Quantification analysis of relative protein expression levels from a and g . ( n  = 4 animals per genotype; two-tailed unpaired t -test). c Tile scan assembly of a coronal section of P14 brain immunostained with anti-P62. Ctx: Cortex; Hip hippocampus and Str Striatum. d Representative images of co-immunostaining analysis with antibodies against P62 (green) and p-Tdp43 (purple) of P14 cortical sections. g Analysis of VPS35 mRNA levels in the frontal lobe of cortex (frontal), hippocampus (hippo), and cerebellum from a cohort of eight unaffected (Control), six FTD-TDP patients with Pgrn mutation, and ten FTD-TDP without Pgrn mutation. (Data were from GEO GSE20141). Expression levels are normalized to mean of the unaffected group (One-way ANOVA with Tukey’s multiple comparison test). Scale bars: in c , 500 μm; in d , 10 μm. Individual data points were shown as dots with group mean ± s.e.m; * P

    Article Snippet: Western blotting Tissues were dissected from both mutant and littermate control mice and lysed with 5× volume per weight 1% Triton X-100 buffer (1% Triton X-100, 20 mM Tris pH 7.4, 150 mM NaCl, 10% glycerol, protease and phosphatase inhibitor cocktail (Millipore)) by homogenization, incubated on ice-water slurry for 20 min, frozen and thawed twice, and ultracentrifuged at 100,000 g , 4 °C for 30 min as described previously [ ] and the supernatant taken as the soluble fraction.

    Techniques: Western Blot, Expressing, Two Tailed Test, Immunostaining, Mutagenesis