triton x 100  (Millipore)


Bioz Verified Symbol Millipore is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99

    Structured Review

    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.
    Triton X 100, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 379 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/triton x 100/product/Millipore
    Average 99 stars, based on 379 article reviews
    Price from $9.99 to $1999.99
    triton x 100 - by Bioz Stars, 2020-04
    99/100 stars

    Images

    1) Product Images from "Membrane Lipid Rafts Are Necessary for the Maintenance of the α7 Nicotinic Acetylcholine Receptor in Somatic Spines of Ciliary Neurons"

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

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.21-02-00504.2001

    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.
    Figure Legend 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.

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

    2) Product Images from "Lack of specificity of antibodies raised against CLN3, the lysosomal/endosomal transmembrane protein mutated in juvenile Batten disease"

    Article Title: Lack of specificity of antibodies raised against CLN3, the lysosomal/endosomal transmembrane protein mutated in juvenile Batten disease

    Journal: Bioscience Reports

    doi: 10.1042/BSR20171229

    Anti-CLN3 antibodies detect the same bands in protein extracts of WT and  Cln3 −/−  mouse embryonic fibroblast cultures Three different anti-CLN3 antibodies [Abnova rabbit anti-CLN3 (D01P), Abnova mouse anti-CLN3 (M03), and Abcam rabbit anti-CLN3 (ab75959)] were tested in immunoblot experiments using cell lysates and isolated membrane proteins from WT and  Cln3 −/−  mouse embryonic fibroblast cultures. Cell lysates were prepared with a lysis buffer containing either 1% Triton X-100 or 1% DDM. Membrane proteins were isolated using the BioVision Membrane Protein Extraction Kit. Sixty micrograms of protein were loaded in each lane of SDS-containing 10% polyacrylamide gels. Prior to loading, samples were incubated at 37°C for 30 min in reducing sample buffer containing 4 M urea. After the electrophoretic separation, proteins were transferred onto nitrocellulose membranes and probed with the anti-CLN3 antibodies (Abnova rabbit, 1:500; Abnova mouse, 1:500; Abcam rabbit, 1:700); M.W. marker: PageRuler Plus (Thermo Fisher Scientific). The immunoblots shown are representative of two separate experiments.
    Figure Legend Snippet: Anti-CLN3 antibodies detect the same bands in protein extracts of WT and Cln3 −/− mouse embryonic fibroblast cultures Three different anti-CLN3 antibodies [Abnova rabbit anti-CLN3 (D01P), Abnova mouse anti-CLN3 (M03), and Abcam rabbit anti-CLN3 (ab75959)] were tested in immunoblot experiments using cell lysates and isolated membrane proteins from WT and Cln3 −/− mouse embryonic fibroblast cultures. Cell lysates were prepared with a lysis buffer containing either 1% Triton X-100 or 1% DDM. Membrane proteins were isolated using the BioVision Membrane Protein Extraction Kit. Sixty micrograms of protein were loaded in each lane of SDS-containing 10% polyacrylamide gels. Prior to loading, samples were incubated at 37°C for 30 min in reducing sample buffer containing 4 M urea. After the electrophoretic separation, proteins were transferred onto nitrocellulose membranes and probed with the anti-CLN3 antibodies (Abnova rabbit, 1:500; Abnova mouse, 1:500; Abcam rabbit, 1:700); M.W. marker: PageRuler Plus (Thermo Fisher Scientific). The immunoblots shown are representative of two separate experiments.

    Techniques Used: Isolation, Lysis, Protein Extraction, Incubation, Marker, Western Blot

    3) Product Images from "Nanoencapsulation of Bacteriophages in Liposomes Prepared Using Microfluidic Hydrodynamic Flow Focusing"

    Article Title: Nanoencapsulation of Bacteriophages in Liposomes Prepared Using Microfluidic Hydrodynamic Flow Focusing

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.02172

    (A) S. aureus  phage K encapsulation in DSPC:cholesterol liposomes. Phages were encapsulated at FRR of 2:1 at room temperature. Phage titer of liposome encapsulated phages prior to and after acid exposure at pH 2.75 (with and without Triton X-100 disruption). *Indicates significance ( p
    Figure Legend Snippet: (A) S. aureus phage K encapsulation in DSPC:cholesterol liposomes. Phages were encapsulated at FRR of 2:1 at room temperature. Phage titer of liposome encapsulated phages prior to and after acid exposure at pH 2.75 (with and without Triton X-100 disruption). *Indicates significance ( p

    Techniques Used:

    E. coli  T3 phage encapsulation in DSPC:cholesterol liposomes. T3 phages were encapsulated at FRR of 2:1 at room temperature. Free phages were separated by centrifugation for 10 min at 13,000 ×  g  and re-suspended in SM buffer, the process was repeated three times to remove unencapsulated phages. Liposomes were disrupted with Triton X-100 and phage titer assessed by plaque assay.  (A)  Phage T3 titers following encapsulation, after removal of unencapsulated free phages (following three wash steps) and after liposome disruption with Triton X-100.  (B)  Phage encapsulation yield using different initial phage titers and after encapsulation, free phage removal by centrifugation (as above) and then liposome disruption with Triton X-100. *Indicates significance ( p
    Figure Legend Snippet: E. coli T3 phage encapsulation in DSPC:cholesterol liposomes. T3 phages were encapsulated at FRR of 2:1 at room temperature. Free phages were separated by centrifugation for 10 min at 13,000 × g and re-suspended in SM buffer, the process was repeated three times to remove unencapsulated phages. Liposomes were disrupted with Triton X-100 and phage titer assessed by plaque assay. (A) Phage T3 titers following encapsulation, after removal of unencapsulated free phages (following three wash steps) and after liposome disruption with Triton X-100. (B) Phage encapsulation yield using different initial phage titers and after encapsulation, free phage removal by centrifugation (as above) and then liposome disruption with Triton X-100. *Indicates significance ( p

    Techniques Used: Centrifugation, Plaque Assay

    4) Product Images from "Membrane Lipid Rafts Are Necessary for the Maintenance of the α7 Nicotinic Acetylcholine Receptor in Somatic Spines of Ciliary Neurons"

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

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.21-02-00504.2001

    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.
    Figure Legend 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.

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

    5) Product Images from "Synthesis and Evaluation of Esterified Hsp70 Agonists in Cellular Models of Protein Aggregation and Folding"

    Article Title: Synthesis and Evaluation of Esterified Hsp70 Agonists in Cellular Models of Protein Aggregation and Folding

    Journal: Bioorganic & medicinal chemistry

    doi: 10.1016/j.bmc.2018.11.011

    Biochemical evaluation of the compounds on α-synuclein aggregation. Cells expressing α-synuclein were treated with compound 1 (grey) or compound 7 (orange) at a final concentration of 100 µM or DMSO (blue) and lysed in a Triton X-100-containing buffer. The amount of α-synuclein was detected by immunoblot analysis and quantified. Data are standardized to the amount of insoluble material in DMSO-treated cells, represent the means from three independent experiments, and are expressed as mean value ± SD. * denotes P
    Figure Legend Snippet: Biochemical evaluation of the compounds on α-synuclein aggregation. Cells expressing α-synuclein were treated with compound 1 (grey) or compound 7 (orange) at a final concentration of 100 µM or DMSO (blue) and lysed in a Triton X-100-containing buffer. The amount of α-synuclein was detected by immunoblot analysis and quantified. Data are standardized to the amount of insoluble material in DMSO-treated cells, represent the means from three independent experiments, and are expressed as mean value ± SD. * denotes P

    Techniques Used: Expressing, Concentration Assay

    6) Product Images from "Splice Variant-Specific Interaction of the NMDA Receptor Subunit NR1 with Neuronal Intermediate Filaments"

    Article Title: Splice Variant-Specific Interaction of the NMDA Receptor Subunit NR1 with Neuronal Intermediate Filaments

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.18-02-00720.1998

    Cofractionation of NF-L and NR1 from rat brain. Synaptic plasma membranes isolated from rat brain (5 μg) were solubilized in either 2% Triton X-100 or 2% SDS, and insoluble proteins were sedimented by centrifugation at 15,000 × g . Proteins
    Figure Legend Snippet: Cofractionation of NF-L and NR1 from rat brain. Synaptic plasma membranes isolated from rat brain (5 μg) were solubilized in either 2% Triton X-100 or 2% SDS, and insoluble proteins were sedimented by centrifugation at 15,000 × g . Proteins

    Techniques Used: Isolation, Centrifugation

    7) Product Images from "Nanoencapsulation of Bacteriophages in Liposomes Prepared Using Microfluidic Hydrodynamic Flow Focusing"

    Article Title: Nanoencapsulation of Bacteriophages in Liposomes Prepared Using Microfluidic Hydrodynamic Flow Focusing

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.02172

    (A) S. aureus  phage K encapsulation in DSPC:cholesterol liposomes. Phages were encapsulated at FRR of 2:1 at room temperature. Phage titer of liposome encapsulated phages prior to and after acid exposure at pH 2.75 (with and without Triton X-100 disruption). *Indicates significance ( p
    Figure Legend Snippet: (A) S. aureus phage K encapsulation in DSPC:cholesterol liposomes. Phages were encapsulated at FRR of 2:1 at room temperature. Phage titer of liposome encapsulated phages prior to and after acid exposure at pH 2.75 (with and without Triton X-100 disruption). *Indicates significance ( p

    Techniques Used:

    E. coli  T3 phage encapsulation in DSPC:cholesterol liposomes. T3 phages were encapsulated at FRR of 2:1 at room temperature. Free phages were separated by centrifugation for 10 min at 13,000 ×  g  and re-suspended in SM buffer, the process was repeated three times to remove unencapsulated phages. Liposomes were disrupted with Triton X-100 and phage titer assessed by plaque assay.  (A)  Phage T3 titers following encapsulation, after removal of unencapsulated free phages (following three wash steps) and after liposome disruption with Triton X-100.  (B)  Phage encapsulation yield using different initial phage titers and after encapsulation, free phage removal by centrifugation (as above) and then liposome disruption with Triton X-100. *Indicates significance ( p
    Figure Legend Snippet: E. coli T3 phage encapsulation in DSPC:cholesterol liposomes. T3 phages were encapsulated at FRR of 2:1 at room temperature. Free phages were separated by centrifugation for 10 min at 13,000 × g and re-suspended in SM buffer, the process was repeated three times to remove unencapsulated phages. Liposomes were disrupted with Triton X-100 and phage titer assessed by plaque assay. (A) Phage T3 titers following encapsulation, after removal of unencapsulated free phages (following three wash steps) and after liposome disruption with Triton X-100. (B) Phage encapsulation yield using different initial phage titers and after encapsulation, free phage removal by centrifugation (as above) and then liposome disruption with Triton X-100. *Indicates significance ( p

    Techniques Used: Centrifugation, Plaque Assay

    8) Product Images from "USP1 (ubiquitin specific peptidase 1) targets ULK1 and regulates its cellular compartmentalization and autophagy"

    Article Title: USP1 (ubiquitin specific peptidase 1) targets ULK1 and regulates its cellular compartmentalization and autophagy

    Journal: Autophagy

    doi: 10.1080/15548627.2018.1535291

    USP1 modulates ULK1 in mammalian cells. (a) U2OS cells were transfected with scrambled control siRNA or 3 different  USP1 -specific siRNAs. The relative amounts of each protein were quantified using ImageJ. The ratio of ULK1:ACTA1 and p-ULK1:ACTA1 was calculated and indicated in the graphs (b and c). (d) HEK-293, U2OS and MCF10AT cells were transfected with scrambled control or  USP1 -specific siRNA. After 72 h, samples were lysed in 1% Triton X-100-containing buffer and the cleared lysates utilized to monitor endogenous ULK1 and ULK1 p-Ser555 protein levels by western blot. The relative amounts of each protein were quantified using ImageJ. The ratio of ULK1:ACTA1 and pULK1:ACTA1 was calculated and indicated in the graphs (e and f). Filled arrow indicates full-length USP1; empty arrow indicates its autocleaved derivative. (g) U2OS cells were transfected with the indicated constructs. After 24 h the samples were lysed in 1% Triton X-100-containing buffer and, the cleared lysates utilized to monitor endogenous ULK1 and ULK1 p-Ser555 protein levels by western blot. The relative amounts of each protein were quantified using ImageJ and the ratio of ULK1:ACTA1 and p-ULK1:ACTA1 was calculated and indicated in the graphs (h and i).
    Figure Legend Snippet: USP1 modulates ULK1 in mammalian cells. (a) U2OS cells were transfected with scrambled control siRNA or 3 different USP1 -specific siRNAs. The relative amounts of each protein were quantified using ImageJ. The ratio of ULK1:ACTA1 and p-ULK1:ACTA1 was calculated and indicated in the graphs (b and c). (d) HEK-293, U2OS and MCF10AT cells were transfected with scrambled control or USP1 -specific siRNA. After 72 h, samples were lysed in 1% Triton X-100-containing buffer and the cleared lysates utilized to monitor endogenous ULK1 and ULK1 p-Ser555 protein levels by western blot. The relative amounts of each protein were quantified using ImageJ. The ratio of ULK1:ACTA1 and pULK1:ACTA1 was calculated and indicated in the graphs (e and f). Filled arrow indicates full-length USP1; empty arrow indicates its autocleaved derivative. (g) U2OS cells were transfected with the indicated constructs. After 24 h the samples were lysed in 1% Triton X-100-containing buffer and, the cleared lysates utilized to monitor endogenous ULK1 and ULK1 p-Ser555 protein levels by western blot. The relative amounts of each protein were quantified using ImageJ and the ratio of ULK1:ACTA1 and p-ULK1:ACTA1 was calculated and indicated in the graphs (h and i).

    Techniques Used: Transfection, Western Blot, Construct

    9) Product Images from "Conserved lipid and small molecule modulation of COQ8 reveals regulation of the ancient kinase-like UbiB family"

    Article Title: Conserved lipid and small molecule modulation of COQ8 reveals regulation of the ancient kinase-like UbiB family

    Journal: Cell chemical biology

    doi: 10.1016/j.chembiol.2017.11.001

    UbiB family members are activated by Triton X-100 and 2-alkylphenols (A) Screen of CoQ intermediate-like compounds with Triton X-100 in an ADP-Glo assay. 2,6-diMeO-HQ, 2,6-dimethoxyhydroquinone; 2-MeO-HQ, 2-methoxyhydroquinone; MeHQ, methylhydroquinone; 4-HB, 4-hydroxybenzoic acid; 3,4-di-HB, 3,4-dihydroxybenzoic acid; 4-MC, 4-methylcatechol; HQ, hydroquinone; 2-MeO-6-MP, 2-methoxy-6-methylphenol; 2-AP, 2-allylphenol; 2-PP, 2-propylphenol; 4-MeOP, 4-methoxyphenol. (B) Malachite green ATPase assay with COQ8A NΔ250  KxGQ mutants, Triton X-100, and 2-alkylphenols. Pi, inorganic phosphate. (C) Drop assay of Δ coq8  transformed with KxGQ mutants. (D) Relative CoQ abundance from Δ coq8  transformed with Coq8p variants. (E) Malachite green ATPase assay of UbiB family members from human, yeast, and  E. coli .
    Figure Legend Snippet: UbiB family members are activated by Triton X-100 and 2-alkylphenols (A) Screen of CoQ intermediate-like compounds with Triton X-100 in an ADP-Glo assay. 2,6-diMeO-HQ, 2,6-dimethoxyhydroquinone; 2-MeO-HQ, 2-methoxyhydroquinone; MeHQ, methylhydroquinone; 4-HB, 4-hydroxybenzoic acid; 3,4-di-HB, 3,4-dihydroxybenzoic acid; 4-MC, 4-methylcatechol; HQ, hydroquinone; 2-MeO-6-MP, 2-methoxy-6-methylphenol; 2-AP, 2-allylphenol; 2-PP, 2-propylphenol; 4-MeOP, 4-methoxyphenol. (B) Malachite green ATPase assay with COQ8A NΔ250 KxGQ mutants, Triton X-100, and 2-alkylphenols. Pi, inorganic phosphate. (C) Drop assay of Δ coq8 transformed with KxGQ mutants. (D) Relative CoQ abundance from Δ coq8 transformed with Coq8p variants. (E) Malachite green ATPase assay of UbiB family members from human, yeast, and E. coli .

    Techniques Used: Glo Assay, ATPase Assay, Transformation Assay

    10) Product Images from "Insertional Inactivation of Treponemadenticola tap1 Results in a Nonmotile Mutant with Elongated Flagellar Hooks"

    Article Title: Insertional Inactivation of Treponemadenticola tap1 Results in a Nonmotile Mutant with Elongated Flagellar Hooks

    Journal: Journal of Bacteriology

    doi:

    Microscopic analysis of the Tap1-deficient mutant JS97. (A) Dark-field micrograph. Bar, 5 μm. (B) Electron micrograph of a JS97 cell showing elongated hooks that do not possess flagellar filaments. The outer sheath was removed by treatment with 1% Triton X-100 reduced as indicated in the text. Bar, 100 nm. (C and D) Electron micrographs of preparations of enriched hooks from JS97. Bars, 100 nm.
    Figure Legend Snippet: Microscopic analysis of the Tap1-deficient mutant JS97. (A) Dark-field micrograph. Bar, 5 μm. (B) Electron micrograph of a JS97 cell showing elongated hooks that do not possess flagellar filaments. The outer sheath was removed by treatment with 1% Triton X-100 reduced as indicated in the text. Bar, 100 nm. (C and D) Electron micrographs of preparations of enriched hooks from JS97. Bars, 100 nm.

    Techniques Used: Mutagenesis

    11) Product Images from "Trimethylamine N-oxide alleviates the severe aggregation and ER stress caused by G98R ?A-crystallin"

    Article Title: Trimethylamine N-oxide alleviates the severe aggregation and ER stress caused by G98R ?A-crystallin

    Journal: Molecular Vision

    doi:

    G98R αA-crystallin was TritonX-100-insoluble and formed aggregates inside cells.  A : Triton X-100 (Tx) solubility assay of wild-type (WT) and cataract-causing mutant CRYAA in B3 cells by western blotting of myc (detecting CRYAA), housekeeping GAPDH, and β-actin. The band densitometry analysis showed the drastic reduction of Tx solubility of G98R CRYAA when compared to WT or other mutants.  B : Direct sequencing of pHis/myc-CRYAA G98R  to indicate the base change at c.292G > A.  C – L : Confocal double immunofluorescence of WT and G98R CRYAA in B3 cells.  C  and  D : A lower magnification to show the expression of WT ( C ) and G98R CRYAA ( D ) in cells.  E – H : G98R CRYAA (myc staining in  F ) formed intracellular aggregates and was intensely co-distributed with PDI ( G ) in the overlay image ( H ).  I – L : WT CRYAA ( J , myc staining) was diffusely distributed in cytoplasm and had only mild co-distribution with PDI ( K ) in overlay image ( L ). Nuclei were stained with DAPI (blue,  E  and  I ). Scale bars: 10 μm ( C – L ). PDI: protein disulphide isomerase; DAPI: 4'-6-diamidino-2-phenylindole.
    Figure Legend Snippet: G98R αA-crystallin was TritonX-100-insoluble and formed aggregates inside cells. A : Triton X-100 (Tx) solubility assay of wild-type (WT) and cataract-causing mutant CRYAA in B3 cells by western blotting of myc (detecting CRYAA), housekeeping GAPDH, and β-actin. The band densitometry analysis showed the drastic reduction of Tx solubility of G98R CRYAA when compared to WT or other mutants. B : Direct sequencing of pHis/myc-CRYAA G98R to indicate the base change at c.292G > A. C – L : Confocal double immunofluorescence of WT and G98R CRYAA in B3 cells. C and D : A lower magnification to show the expression of WT ( C ) and G98R CRYAA ( D ) in cells. E – H : G98R CRYAA (myc staining in F ) formed intracellular aggregates and was intensely co-distributed with PDI ( G ) in the overlay image ( H ). I – L : WT CRYAA ( J , myc staining) was diffusely distributed in cytoplasm and had only mild co-distribution with PDI ( K ) in overlay image ( L ). Nuclei were stained with DAPI (blue, E and I ). Scale bars: 10 μm ( C – L ). PDI: protein disulphide isomerase; DAPI: 4'-6-diamidino-2-phenylindole.

    Techniques Used: Solubility, Mutagenesis, Western Blot, Sequencing, Immunofluorescence, Expressing, Staining

    12) Product Images from "Membrane Lipid Rafts Are Necessary for the Maintenance of the α7 Nicotinic Acetylcholine Receptor in Somatic Spines of Ciliary Neurons"

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

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.21-02-00504.2001

    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.
    Figure Legend 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.

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

    13) Product Images from "Membrane Lipid Rafts Are Necessary for the Maintenance of the α7 Nicotinic Acetylcholine Receptor in Somatic Spines of Ciliary Neurons"

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

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.21-02-00504.2001

    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.
    Figure Legend 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.

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

    14) Product Images from "Membrane Lipid Rafts Are Necessary for the Maintenance of the α7 Nicotinic Acetylcholine Receptor in Somatic Spines of Ciliary Neurons"

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

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.21-02-00504.2001

    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.
    Figure Legend 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.

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

    Related Articles

    Centrifugation:

    Article Title: Trimethylamine N-oxide alleviates the severe aggregation and ER stress caused by G98R ?A-crystallin
    Article Snippet: After centrifugation, the supernatant was collected and denatured in sample buffer with a final concentration of 2% sodium dodecyl sulfate (SDS; BioRad, Hercules, CA) and 50 mM DTT (Sigma). .. For Triton X-100 (Tx) solubility analysis, cells were washed twice with ice-cold PBS and added to lysis buffer, which contained 100 mM Tris-HCl (pH 7.4), 3 mM ethylene glycol tetraacetic acid (EGTA; Sigma), 5 mM MgCl2 , 0.5% Triton X-100 (Tx; Sigma), protease inhibitor cocktail, and 1 mM PMSF at 5×106 cells/ml, for 2 min on ice.

    Article Title: USP1 (ubiquitin specific peptidase 1) targets ULK1 and regulates its cellular compartmentalization and autophagy
    Article Snippet: Western blotting was performed on cells protein lysates (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100 (Sigma-Aldrich, T8787), 0.5 mM NaF, 1 mM sodium orthovanadate (Sigma-Aldrich, S6508), complete Protease Inhibitor Cocktail [Sigma-Aldrich, P2714]). .. Lysates were clarified by centrifugation for 15 min at 4°C and protein concentrations were assessed using the Bradford protein assay (Bio-Rad Laboratories, 5000001).

    Blocking Assay:

    Article Title: Synthesis and Evaluation of Esterified Hsp70 Agonists in Cellular Models of Protein Aggregation and Folding
    Article Snippet: Twenty-four hours after treatment, cells were washed with PBS and fixed with 4% PFA for 15 minutes at room temperature (RT), followed by a permeabilization step with 0.1% Triton X-100 (Sigma Aldrich, St. Louis, MO, USA) for 20 minutes at RT. .. After blocking in 1.5% BSA (NZYTECH in PBS for 1 h, cells were incubated with primary antibody.

    Incubation:

    Article Title: Lack of specificity of antibodies raised against CLN3, the lysosomal/endosomal transmembrane protein mutated in juvenile Batten disease
    Article Snippet: .. The cell pellets were suspended in ice-cold lysis buffer containing either DDM [50 mM sodium phosphate (pH 7.4), 1% DDM, 1 mM DTT, protease inhibitor cocktail, and phosphatase inhibitor cocktail (Sigma)] or Triton X-100 [50 mM Tris/HCl (pH 7.5), 300 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1 mM DTT, protease inhibitor cocktail, and phosphatase inhibitor cocktail (Sigma)], vortexed vigorously for 10 s, and incubated on ice for 30 min. .. The supernatants were transferred into new precooled 1.5-ml microtubes and total protein concentration of the lysates was determined by the Pierce 660-nm protein assay (Pierce, Rockford, IL).

    Article Title: Conserved lipid and small molecule modulation of COQ8 reveals regulation of the ancient kinase-like UbiB family
    Article Snippet: Coq8pNΔ41 (1 μM), COQ8ANΔ250 (2 or 4 μM), COQ8ANΔ162 (2 μM), or MBP-UbiBCΔ47 (0.5 μM) were mixed with liposomes (~3.33 mM), ATP (100 μM), MgCl2 (4 mM), CMK (20 μM) or DMSO (0.2% v/v), Triton X-100 (1 mM) or reduced Triton X-100 (1 mM) (Sigma Aldrich) and CoQ headgroup analogs (Sigma Aldrich) dissolved as 200 mM stock solutions in DMSO (2-PP, 2-propylphenol; 2-MeO-6-MP, 2-methoxy-6-methylphenol; 4-methylcatechol; 4-HB, 4-hydroxybenzoic acid; 2-MeO-HQ, 2-methoxyhydroquinone; 4-MeOP, 4-methoxyphenol; 2-AP, 2-allylphenol; HQ, hydroquinone; 3,4-di-HB, 3,4-dihydroxybenzoic acid; MeHQ, methylhydroquinone; 2,6-diMeO-HQ, 2,6-dimethoxyhydroquinone) (1 mM) (final concentrations for reaction components). .. Then ADP-Glo Reagent (5 μL) was added and incubated (40 min, r.t. [~21 °C], covered).

    Article Title: Insertional Inactivation of Treponemadenticola tap1 Results in a Nonmotile Mutant with Elongated Flagellar Hooks
    Article Snippet: .. The pellet was resuspended in the same amount of water containing 1% Triton X-100 reduced, a chemically modified form of Triton X-100 (Aldrich Chemical Co., Milwaukee, Wis.), and incubated overnight at 4°C. ..

    Article Title: Synthesis and Evaluation of Esterified Hsp70 Agonists in Cellular Models of Protein Aggregation and Folding
    Article Snippet: Twenty-four hours after treatment, cells were washed with PBS and fixed with 4% PFA for 15 minutes at room temperature (RT), followed by a permeabilization step with 0.1% Triton X-100 (Sigma Aldrich, St. Louis, MO, USA) for 20 minutes at RT. .. After blocking in 1.5% BSA (NZYTECH in PBS for 1 h, cells were incubated with primary antibody.

    Article Title: USP1 (ubiquitin specific peptidase 1) targets ULK1 and regulates its cellular compartmentalization and autophagy
    Article Snippet: Western blotting was performed on cells protein lysates (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100 (Sigma-Aldrich, T8787), 0.5 mM NaF, 1 mM sodium orthovanadate (Sigma-Aldrich, S6508), complete Protease Inhibitor Cocktail [Sigma-Aldrich, P2714]). .. The membranes were blocked with 5% non-fat dry milk at room temperature for 1 h on a rotary shaker, followed by overnight incubation at 4°C with appropriate antibodies, incubated with horseradish peroxidase-conjugated secondary antibodies, and analyzed by enhanced chemiluminescence.

    HAT Assay:

    Article Title: Synthesis and Evaluation of Esterified Hsp70 Agonists in Cellular Models of Protein Aggregation and Folding
    Article Snippet: Twenty-four hours after treatment, cells were washed with PBS and fixed with 4% PFA for 15 minutes at room temperature (RT), followed by a permeabilization step with 0.1% Triton X-100 (Sigma Aldrich, St. Louis, MO, USA) for 20 minutes at RT. .. Primary antibodies used were: rabbit anti-ASYN (1:2000, Santa Cruz Biotechnology) and mouse anti-V5 (1:2000, Life Technologies-Invitrogen) overnight at 4 °C, followed by rinsing with PBS and incubation for 2 hat RT with the following secondary antibodies: Alexa fluor 488 donkey anti-rabbit (1:2000, Life Technologies-Invitrogen) and Alexa Fluor 555 donkey anti-mouse (1:2000, Life Technologies-Invitrogen).

    Modification:

    Article Title: Insertional Inactivation of Treponemadenticola tap1 Results in a Nonmotile Mutant with Elongated Flagellar Hooks
    Article Snippet: .. The pellet was resuspended in the same amount of water containing 1% Triton X-100 reduced, a chemically modified form of Triton X-100 (Aldrich Chemical Co., Milwaukee, Wis.), and incubated overnight at 4°C. ..

    Western Blot:

    Article Title: Trimethylamine N-oxide alleviates the severe aggregation and ER stress caused by G98R ?A-crystallin
    Article Snippet: For Triton X-100 (Tx) solubility analysis, cells were washed twice with ice-cold PBS and added to lysis buffer, which contained 100 mM Tris-HCl (pH 7.4), 3 mM ethylene glycol tetraacetic acid (EGTA; Sigma), 5 mM MgCl2 , 0.5% Triton X-100 (Tx; Sigma), protease inhibitor cocktail, and 1 mM PMSF at 5×106 cells/ml, for 2 min on ice. .. The samples were analyzed with 10% SDS-polyacrylamide gel electrophoresis and western blotted using horseradish peroxidase (HRP)-conjugated antibodies against myc (BD Biosciences, San Jose, CA) recognizing CRYAA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Sigma), or β-actin (Sigma), or monoclonal antibodies against binding immunoglobulin protein (BiP; BD BioSciences), C/EBP homologous protein/growth arrest and DNA damage-inducible gene 153 (CHOP/GADD153; Santa Cruz Biotech, Santa Cruz, CA), caspase-3 (Santa Cruz Biotech), or phosphorylated ER kinase (PERK; Santa Cruz Biotech), followed by appropriate HRP-conjugated immunoglobulin (Ig) secondary antibodies.

    Article Title: USP1 (ubiquitin specific peptidase 1) targets ULK1 and regulates its cellular compartmentalization and autophagy
    Article Snippet: .. Western blotting was performed on cells protein lysates (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100 (Sigma-Aldrich, T8787), 0.5 mM NaF, 1 mM sodium orthovanadate (Sigma-Aldrich, S6508), complete Protease Inhibitor Cocktail [Sigma-Aldrich, P2714]). .. Lysates were clarified by centrifugation for 15 min at 4°C and protein concentrations were assessed using the Bradford protein assay (Bio-Rad Laboratories, 5000001).

    Article Title: Membrane Lipid Rafts Are Necessary for the Maintenance of the α7 Nicotinic Acetylcholine Receptor in Somatic Spines of Ciliary Neurons
    Article Snippet: .. Thereafter, the membrane-containing fractions were extracted with Triton X-100 at 4°C, separated by density gradient centrifugation, and analyzed by SDS-PAGE and Western blotting. ..

    Electron Microscopy:

    Article Title: Insertional Inactivation of Treponemadenticola tap1 Results in a Nonmotile Mutant with Elongated Flagellar Hooks
    Article Snippet: Paragraph title: Isolation of flagellar hooks and electron microscopy. ... The pellet was resuspended in the same amount of water containing 1% Triton X-100 reduced, a chemically modified form of Triton X-100 (Aldrich Chemical Co., Milwaukee, Wis.), and incubated overnight at 4°C.

    Transfection:

    Article Title: Synthesis and Evaluation of Esterified Hsp70 Agonists in Cellular Models of Protein Aggregation and Folding
    Article Snippet: Sixteen hours after transfection, H4 cells were fed with fresh medium and treated either with the Hsp70 agonists DWN-72–323 (compound 7) or MAL1–271 (compound 1 ) at final concentrations of 1, 10 and 100 µM. .. Twenty-four hours after treatment, cells were washed with PBS and fixed with 4% PFA for 15 minutes at room temperature (RT), followed by a permeabilization step with 0.1% Triton X-100 (Sigma Aldrich, St. Louis, MO, USA) for 20 minutes at RT.

    Concentration Assay:

    Article Title: Insertional Inactivation of Treponemadenticola tap1 Results in a Nonmotile Mutant with Elongated Flagellar Hooks
    Article Snippet: Next, Triton X-100 was added to a final concentration of 2% and the incubation was continued for 1 h at 20°C. .. The pellet was resuspended in the same amount of water containing 1% Triton X-100 reduced, a chemically modified form of Triton X-100 (Aldrich Chemical Co., Milwaukee, Wis.), and incubated overnight at 4°C.

    Article Title: Trimethylamine N-oxide alleviates the severe aggregation and ER stress caused by G98R ?A-crystallin
    Article Snippet: After centrifugation, the supernatant was collected and denatured in sample buffer with a final concentration of 2% sodium dodecyl sulfate (SDS; BioRad, Hercules, CA) and 50 mM DTT (Sigma). .. For Triton X-100 (Tx) solubility analysis, cells were washed twice with ice-cold PBS and added to lysis buffer, which contained 100 mM Tris-HCl (pH 7.4), 3 mM ethylene glycol tetraacetic acid (EGTA; Sigma), 5 mM MgCl2 , 0.5% Triton X-100 (Tx; Sigma), protease inhibitor cocktail, and 1 mM PMSF at 5×106 cells/ml, for 2 min on ice.

    Protease Inhibitor:

    Article Title: Lack of specificity of antibodies raised against CLN3, the lysosomal/endosomal transmembrane protein mutated in juvenile Batten disease
    Article Snippet: .. The cell pellets were suspended in ice-cold lysis buffer containing either DDM [50 mM sodium phosphate (pH 7.4), 1% DDM, 1 mM DTT, protease inhibitor cocktail, and phosphatase inhibitor cocktail (Sigma)] or Triton X-100 [50 mM Tris/HCl (pH 7.5), 300 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1 mM DTT, protease inhibitor cocktail, and phosphatase inhibitor cocktail (Sigma)], vortexed vigorously for 10 s, and incubated on ice for 30 min. .. The supernatants were transferred into new precooled 1.5-ml microtubes and total protein concentration of the lysates was determined by the Pierce 660-nm protein assay (Pierce, Rockford, IL).

    Article Title: Trimethylamine N-oxide alleviates the severe aggregation and ER stress caused by G98R ?A-crystallin
    Article Snippet: .. For Triton X-100 (Tx) solubility analysis, cells were washed twice with ice-cold PBS and added to lysis buffer, which contained 100 mM Tris-HCl (pH 7.4), 3 mM ethylene glycol tetraacetic acid (EGTA; Sigma), 5 mM MgCl2 , 0.5% Triton X-100 (Tx; Sigma), protease inhibitor cocktail, and 1 mM PMSF at 5×106 cells/ml, for 2 min on ice. .. After centrifugation, the supernatant containing Tx-soluble protein was denatured in SDS buffer.

    Article Title: USP1 (ubiquitin specific peptidase 1) targets ULK1 and regulates its cellular compartmentalization and autophagy
    Article Snippet: .. Western blotting was performed on cells protein lysates (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100 (Sigma-Aldrich, T8787), 0.5 mM NaF, 1 mM sodium orthovanadate (Sigma-Aldrich, S6508), complete Protease Inhibitor Cocktail [Sigma-Aldrich, P2714]). .. Lysates were clarified by centrifugation for 15 min at 4°C and protein concentrations were assessed using the Bradford protein assay (Bio-Rad Laboratories, 5000001).

    Solubility:

    Article Title: Trimethylamine N-oxide alleviates the severe aggregation and ER stress caused by G98R ?A-crystallin
    Article Snippet: .. For Triton X-100 (Tx) solubility analysis, cells were washed twice with ice-cold PBS and added to lysis buffer, which contained 100 mM Tris-HCl (pH 7.4), 3 mM ethylene glycol tetraacetic acid (EGTA; Sigma), 5 mM MgCl2 , 0.5% Triton X-100 (Tx; Sigma), protease inhibitor cocktail, and 1 mM PMSF at 5×106 cells/ml, for 2 min on ice. .. After centrifugation, the supernatant containing Tx-soluble protein was denatured in SDS buffer.

    Protein Concentration:

    Article Title: Lack of specificity of antibodies raised against CLN3, the lysosomal/endosomal transmembrane protein mutated in juvenile Batten disease
    Article Snippet: The cell pellets were suspended in ice-cold lysis buffer containing either DDM [50 mM sodium phosphate (pH 7.4), 1% DDM, 1 mM DTT, protease inhibitor cocktail, and phosphatase inhibitor cocktail (Sigma)] or Triton X-100 [50 mM Tris/HCl (pH 7.5), 300 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1 mM DTT, protease inhibitor cocktail, and phosphatase inhibitor cocktail (Sigma)], vortexed vigorously for 10 s, and incubated on ice for 30 min. .. The supernatants were transferred into new precooled 1.5-ml microtubes and total protein concentration of the lysates was determined by the Pierce 660-nm protein assay (Pierce, Rockford, IL).

    Sonication:

    Article Title: Trimethylamine N-oxide alleviates the severe aggregation and ER stress caused by G98R ?A-crystallin
    Article Snippet: For Triton X-100 (Tx) solubility analysis, cells were washed twice with ice-cold PBS and added to lysis buffer, which contained 100 mM Tris-HCl (pH 7.4), 3 mM ethylene glycol tetraacetic acid (EGTA; Sigma), 5 mM MgCl2 , 0.5% Triton X-100 (Tx; Sigma), protease inhibitor cocktail, and 1 mM PMSF at 5×106 cells/ml, for 2 min on ice. .. The pellet containing Tx-insoluble protein was washed twice with ice-cold PBS, sonicated, and denatured in urea-SDS buffer [ ].

    Binding Assay:

    Article Title: Trimethylamine N-oxide alleviates the severe aggregation and ER stress caused by G98R ?A-crystallin
    Article Snippet: For Triton X-100 (Tx) solubility analysis, cells were washed twice with ice-cold PBS and added to lysis buffer, which contained 100 mM Tris-HCl (pH 7.4), 3 mM ethylene glycol tetraacetic acid (EGTA; Sigma), 5 mM MgCl2 , 0.5% Triton X-100 (Tx; Sigma), protease inhibitor cocktail, and 1 mM PMSF at 5×106 cells/ml, for 2 min on ice. .. The samples were analyzed with 10% SDS-polyacrylamide gel electrophoresis and western blotted using horseradish peroxidase (HRP)-conjugated antibodies against myc (BD Biosciences, San Jose, CA) recognizing CRYAA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Sigma), or β-actin (Sigma), or monoclonal antibodies against binding immunoglobulin protein (BiP; BD BioSciences), C/EBP homologous protein/growth arrest and DNA damage-inducible gene 153 (CHOP/GADD153; Santa Cruz Biotech, Santa Cruz, CA), caspase-3 (Santa Cruz Biotech), or phosphorylated ER kinase (PERK; Santa Cruz Biotech), followed by appropriate HRP-conjugated immunoglobulin (Ig) secondary antibodies.

    Nucleic Acid Electrophoresis:

    Article Title: Trimethylamine N-oxide alleviates the severe aggregation and ER stress caused by G98R ?A-crystallin
    Article Snippet: For Triton X-100 (Tx) solubility analysis, cells were washed twice with ice-cold PBS and added to lysis buffer, which contained 100 mM Tris-HCl (pH 7.4), 3 mM ethylene glycol tetraacetic acid (EGTA; Sigma), 5 mM MgCl2 , 0.5% Triton X-100 (Tx; Sigma), protease inhibitor cocktail, and 1 mM PMSF at 5×106 cells/ml, for 2 min on ice. .. The samples were analyzed with 10% SDS-polyacrylamide gel electrophoresis and western blotted using horseradish peroxidase (HRP)-conjugated antibodies against myc (BD Biosciences, San Jose, CA) recognizing CRYAA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Sigma), or β-actin (Sigma), or monoclonal antibodies against binding immunoglobulin protein (BiP; BD BioSciences), C/EBP homologous protein/growth arrest and DNA damage-inducible gene 153 (CHOP/GADD153; Santa Cruz Biotech, Santa Cruz, CA), caspase-3 (Santa Cruz Biotech), or phosphorylated ER kinase (PERK; Santa Cruz Biotech), followed by appropriate HRP-conjugated immunoglobulin (Ig) secondary antibodies.

    Isolation:

    Article Title: Insertional Inactivation of Treponemadenticola tap1 Results in a Nonmotile Mutant with Elongated Flagellar Hooks
    Article Snippet: Paragraph title: Isolation of flagellar hooks and electron microscopy. ... The pellet was resuspended in the same amount of water containing 1% Triton X-100 reduced, a chemically modified form of Triton X-100 (Aldrich Chemical Co., Milwaukee, Wis.), and incubated overnight at 4°C.

    Transmission Assay:

    Article Title: Insertional Inactivation of Treponemadenticola tap1 Results in a Nonmotile Mutant with Elongated Flagellar Hooks
    Article Snippet: The pellet was resuspended in the same amount of water containing 1% Triton X-100 reduced, a chemically modified form of Triton X-100 (Aldrich Chemical Co., Milwaukee, Wis.), and incubated overnight at 4°C. .. After the final wash, excess water was removed, the grids were briefly floated on 2% sodium phosphotungstate (pH 7.0), liquid was removed by wicking, and the samples were viewed in a Zeiss (LEO) 910 transmission electron microscope operating at 80 keV.

    Microscopy:

    Article Title: Insertional Inactivation of Treponemadenticola tap1 Results in a Nonmotile Mutant with Elongated Flagellar Hooks
    Article Snippet: The pellet was resuspended in the same amount of water containing 1% Triton X-100 reduced, a chemically modified form of Triton X-100 (Aldrich Chemical Co., Milwaukee, Wis.), and incubated overnight at 4°C. .. After the final wash, excess water was removed, the grids were briefly floated on 2% sodium phosphotungstate (pH 7.0), liquid was removed by wicking, and the samples were viewed in a Zeiss (LEO) 910 transmission electron microscope operating at 80 keV.

    Purification:

    Article Title: Nanoencapsulation of Bacteriophages in Liposomes Prepared Using Microfluidic Hydrodynamic Flow Focusing
    Article Snippet: Phospholipid DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) was purchased in dry powder form from BACHEM (Bubendorf, Switzerland) and was used without any further purification. .. Cholesterol ≥99%, Chloroform anhydrous ≥99%, Isopropanol anhydrous 99.5%, and Triton X-100 were purchased from Sigma-Aldrich, UK.

    Bradford Protein Assay:

    Article Title: USP1 (ubiquitin specific peptidase 1) targets ULK1 and regulates its cellular compartmentalization and autophagy
    Article Snippet: Western blotting was performed on cells protein lysates (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100 (Sigma-Aldrich, T8787), 0.5 mM NaF, 1 mM sodium orthovanadate (Sigma-Aldrich, S6508), complete Protease Inhibitor Cocktail [Sigma-Aldrich, P2714]). .. Lysates were clarified by centrifugation for 15 min at 4°C and protein concentrations were assessed using the Bradford protein assay (Bio-Rad Laboratories, 5000001).

    Lysis:

    Article Title: Lack of specificity of antibodies raised against CLN3, the lysosomal/endosomal transmembrane protein mutated in juvenile Batten disease
    Article Snippet: .. The cell pellets were suspended in ice-cold lysis buffer containing either DDM [50 mM sodium phosphate (pH 7.4), 1% DDM, 1 mM DTT, protease inhibitor cocktail, and phosphatase inhibitor cocktail (Sigma)] or Triton X-100 [50 mM Tris/HCl (pH 7.5), 300 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1 mM DTT, protease inhibitor cocktail, and phosphatase inhibitor cocktail (Sigma)], vortexed vigorously for 10 s, and incubated on ice for 30 min. .. The supernatants were transferred into new precooled 1.5-ml microtubes and total protein concentration of the lysates was determined by the Pierce 660-nm protein assay (Pierce, Rockford, IL).

    Article Title: Trimethylamine N-oxide alleviates the severe aggregation and ER stress caused by G98R ?A-crystallin
    Article Snippet: .. For Triton X-100 (Tx) solubility analysis, cells were washed twice with ice-cold PBS and added to lysis buffer, which contained 100 mM Tris-HCl (pH 7.4), 3 mM ethylene glycol tetraacetic acid (EGTA; Sigma), 5 mM MgCl2 , 0.5% Triton X-100 (Tx; Sigma), protease inhibitor cocktail, and 1 mM PMSF at 5×106 cells/ml, for 2 min on ice. .. After centrifugation, the supernatant containing Tx-soluble protein was denatured in SDS buffer.

    SDS Page:

    Article Title: USP1 (ubiquitin specific peptidase 1) targets ULK1 and regulates its cellular compartmentalization and autophagy
    Article Snippet: Western blotting was performed on cells protein lysates (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100 (Sigma-Aldrich, T8787), 0.5 mM NaF, 1 mM sodium orthovanadate (Sigma-Aldrich, S6508), complete Protease Inhibitor Cocktail [Sigma-Aldrich, P2714]). .. Samples containing equal amounts of protein were boiled in SDS sample buffer, resolved using SDS-PAGE (25 μg protein per lane) and transferred to nitrocellulose membranes.

    Article Title: Membrane Lipid Rafts Are Necessary for the Maintenance of the α7 Nicotinic Acetylcholine Receptor in Somatic Spines of Ciliary Neurons
    Article Snippet: .. Thereafter, the membrane-containing fractions were extracted with Triton X-100 at 4°C, separated by density gradient centrifugation, and analyzed by SDS-PAGE and Western blotting. ..

    Quantitation Assay:

    Article Title: Synthesis and Evaluation of Esterified Hsp70 Agonists in Cellular Models of Protein Aggregation and Folding
    Article Snippet: Paragraph title: 2.3. Quantitation of α-synuclein aggregation in a mammalian cell model ... Twenty-four hours after treatment, cells were washed with PBS and fixed with 4% PFA for 15 minutes at room temperature (RT), followed by a permeabilization step with 0.1% Triton X-100 (Sigma Aldrich, St. Louis, MO, USA) for 20 minutes at RT.

    Immunoprecipitation:

    Article Title: USP1 (ubiquitin specific peptidase 1) targets ULK1 and regulates its cellular compartmentalization and autophagy
    Article Snippet: Paragraph title: Western blot analysis and immunoprecipitation ... Western blotting was performed on cells protein lysates (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100 (Sigma-Aldrich, T8787), 0.5 mM NaF, 1 mM sodium orthovanadate (Sigma-Aldrich, S6508), complete Protease Inhibitor Cocktail [Sigma-Aldrich, P2714]).

    Staining:

    Article Title: Synthesis and Evaluation of Esterified Hsp70 Agonists in Cellular Models of Protein Aggregation and Folding
    Article Snippet: Twenty-four hours after treatment, cells were washed with PBS and fixed with 4% PFA for 15 minutes at room temperature (RT), followed by a permeabilization step with 0.1% Triton X-100 (Sigma Aldrich, St. Louis, MO, USA) for 20 minutes at RT. .. Finally, cells were stained with DAPI (1:5000 in PBS) for 5 min and maintained in PBS for epifluorescence microscopy.

    Gradient Centrifugation:

    Article Title: Membrane Lipid Rafts Are Necessary for the Maintenance of the α7 Nicotinic Acetylcholine Receptor in Somatic Spines of Ciliary Neurons
    Article Snippet: .. Thereafter, the membrane-containing fractions were extracted with Triton X-100 at 4°C, separated by density gradient centrifugation, and analyzed by SDS-PAGE and Western blotting. ..

    Epifluorescence Microscopy:

    Article Title: Synthesis and Evaluation of Esterified Hsp70 Agonists in Cellular Models of Protein Aggregation and Folding
    Article Snippet: Twenty-four hours after treatment, cells were washed with PBS and fixed with 4% PFA for 15 minutes at room temperature (RT), followed by a permeabilization step with 0.1% Triton X-100 (Sigma Aldrich, St. Louis, MO, USA) for 20 minutes at RT. .. Finally, cells were stained with DAPI (1:5000 in PBS) for 5 min and maintained in PBS for epifluorescence microscopy.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    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 187 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/immunoprecipitation buffer/product/Millipore
    Average 99 stars, based on 187 article reviews
    Price from $9.99 to $1999.99
    immunoprecipitation buffer - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    99
    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.
    Triton X 100, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 379 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/triton x 100/product/Millipore
    Average 99 stars, based on 379 article reviews
    Price from $9.99 to $1999.99
    triton x 100 - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    Image Search Results


    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

    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: The samples were then washed three times with solubilization solution, twice with of 0.1 m phosphate buffer, pH 7.3, 0.5% Triton X-100, and 1 m NaCl, and twice with 0.1 m phosphate buffer, pH 7.3, and 0.5% Triton X-100 and resuspended in 50 μl of Laemmli sample buffer.

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

    BACE1 and Cdk5/p35/p25 pathway components are increased in the brains of AD patients and the 5XFAD mouse model of AD. A,  brain samples from 13 noncognitively impaired controls ( NCI ) and nine Alzheimer patients ( AD ) were homogenized in PBS, 1% Triton X-100 and 15 μg of protein per lane were analyzed by immunoblot. Signals were normalized across blots using duplicate samples run on both gels (samples 17 and 18 for BACE1 and samples 10, 17, 18, and 19 for Cdk5 and p25/p35). BACE1 and Cdk5 signals were first normalized across blots and then were finally normalized using actin signals. The p25 and p35 signals were normalized across blots and then the p25:p35 ratio was calculated directly. The doublet at 35 kDa in  A ). We also observed a nonspecific band (labeled  NS  in  A ) near 25 kDa.  B  and  C , Cdk5 levels were elevated in AD patients ( B ) and correlated significantly with BACE1 levels ( C ).  AU,  arbitrary units.  D,  there was a small but significant increase in the ratio of p25/p35, which could also contribute to elevated Cdk5 activity.  E,  hemibrains from 2-month-old 5XFAD mice and nontransgenic littermate controls were homogenized in PBS, 1% Triton X-100, and 15 μg of protein were analyzed by immunoblot. BACE1 and Cdk5 signals were normalized using actin signals. Levels of BACE1 and Cdk5 were elevated in 5XFAD brains, as were those of both p25 and p35.  Error bars  = mean ± S.E.; *,  p  ≤ 0.05; **,  p  ≤ 0.01; ***,  p  ≤ 0.001.

    Journal: The Journal of Biological Chemistry

    Article Title: Cdk5 Protein Inhibition and A?42 Increase BACE1 Protein Level in Primary Neurons by a Post-transcriptional Mechanism

    doi: 10.1074/jbc.M111.333914

    Figure Lengend Snippet: BACE1 and Cdk5/p35/p25 pathway components are increased in the brains of AD patients and the 5XFAD mouse model of AD. A, brain samples from 13 noncognitively impaired controls ( NCI ) and nine Alzheimer patients ( AD ) were homogenized in PBS, 1% Triton X-100 and 15 μg of protein per lane were analyzed by immunoblot. Signals were normalized across blots using duplicate samples run on both gels (samples 17 and 18 for BACE1 and samples 10, 17, 18, and 19 for Cdk5 and p25/p35). BACE1 and Cdk5 signals were first normalized across blots and then were finally normalized using actin signals. The p25 and p35 signals were normalized across blots and then the p25:p35 ratio was calculated directly. The doublet at 35 kDa in A ). We also observed a nonspecific band (labeled NS in A ) near 25 kDa. B and C , Cdk5 levels were elevated in AD patients ( B ) and correlated significantly with BACE1 levels ( C ). AU, arbitrary units. D, there was a small but significant increase in the ratio of p25/p35, which could also contribute to elevated Cdk5 activity. E, hemibrains from 2-month-old 5XFAD mice and nontransgenic littermate controls were homogenized in PBS, 1% Triton X-100, and 15 μg of protein were analyzed by immunoblot. BACE1 and Cdk5 signals were normalized using actin signals. Levels of BACE1 and Cdk5 were elevated in 5XFAD brains, as were those of both p25 and p35. Error bars = mean ± S.E.; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.

    Article Snippet: Frozen tissues (0.2–0.4 g) were homogenized in 1× PBS with 1% Triton X-100, supplemented with protease inhibitors (Calbiochem) and Halt Phosphatase Inhibitor Mixture (Thermo Scientific).

    Techniques: Labeling, Activity Assay, Mouse Assay

    Binding of hN3-B3 S  antibody to tethered NTPDase3 mutants. ( A ) Plates with immobilized NTPDase3 mutants were incubated with hN3-B3 S  monoclonal antibody. The bound antibody was detected using the secondary goat anti-mouse HRP-conjugated antibody and a chemiluminescence reaction. The signal in three replicate wells is shown for each mutant. ( B ) Quantitative estimate of the bound hN3-B3 S  shown in panel (A). Chemiluminescence intensity was determined using AlphaEaseFC software (Alpha Innotech) and expressed in arbitrary units, where one unit corresponds to the intensity of the S87C mutant. The background intensity of C10S is approximately the same as in wells where the membranes of COS cells transfected with an empty vector (pcDNA3) were used, and considered to be zero. The numbers represent means of three wells. ( C ) Susceptibility of cysteine mutants for biotinylation. Proteins were extracted from biotinylated cell membranes with Triton X-100 and incubated with streptavidin agarose beads. Proteins bound to beads (shown in lanes B) were eluted by SDS and analyzed together with non-bound proteins (shown in lanes N) by western blotting using the polyclonal KLH11 antibody for NTPDase3 detection. The amount of NTPDase3 in lane ‘B’ indicates the efficiency of biotinylation. ( D ) Qualitative estimate of the amount of NTPDase3 immobilized on streptavidin plates. The presence of biotinylated NTPDase3 (lanes ‘B’ in Panel C) implies that a certain amount of this protein is immobilized on streptavidin plates (denoted by ‘+’ in Panel D). The lack of biotinylation (no NTPDase3 is detected in lanes ‘B’, Panel C) implies that little or no NTPDase3 immobilized on plates (marked with ‘0’ in Panel D). ( E ) Qualitative estimation of the ratio of the amount of hN3-B3 S  antibody bound to plates (chemiluminescence intensity, Panel B) to the amount of NTPDase3 immobilized on plates (Panel D). Values above zero, zero and undefined are denoted by ‘+’, ‘0’ and ‘0/0’, respectively. ( F ) Antibody binding to immobilized NTPDase3 (denoted by ‘+’, Panel E) suggests epitope accessibility (denoted by ‘a’ in Panel F). The lack of antibody binding to immobilized enzyme (marked with ‘0’ in Panel E) suggests that the epitope is masked (marked with ‘M’ in Panel F). The 0/0 ratio for mutants A267C and D414C (Panel E) makes the evaluation of epitope accessibility uncertain.

    Journal: Protein Engineering, Design and Selection

    Article Title: Epitope mapping in cell surface proteins by site-directed masking: defining the structural elements of NTPDase3 inhibition by a monoclonal antibody

    doi: 10.1093/protein/gzq027

    Figure Lengend Snippet: Binding of hN3-B3 S antibody to tethered NTPDase3 mutants. ( A ) Plates with immobilized NTPDase3 mutants were incubated with hN3-B3 S monoclonal antibody. The bound antibody was detected using the secondary goat anti-mouse HRP-conjugated antibody and a chemiluminescence reaction. The signal in three replicate wells is shown for each mutant. ( B ) Quantitative estimate of the bound hN3-B3 S shown in panel (A). Chemiluminescence intensity was determined using AlphaEaseFC software (Alpha Innotech) and expressed in arbitrary units, where one unit corresponds to the intensity of the S87C mutant. The background intensity of C10S is approximately the same as in wells where the membranes of COS cells transfected with an empty vector (pcDNA3) were used, and considered to be zero. The numbers represent means of three wells. ( C ) Susceptibility of cysteine mutants for biotinylation. Proteins were extracted from biotinylated cell membranes with Triton X-100 and incubated with streptavidin agarose beads. Proteins bound to beads (shown in lanes B) were eluted by SDS and analyzed together with non-bound proteins (shown in lanes N) by western blotting using the polyclonal KLH11 antibody for NTPDase3 detection. The amount of NTPDase3 in lane ‘B’ indicates the efficiency of biotinylation. ( D ) Qualitative estimate of the amount of NTPDase3 immobilized on streptavidin plates. The presence of biotinylated NTPDase3 (lanes ‘B’ in Panel C) implies that a certain amount of this protein is immobilized on streptavidin plates (denoted by ‘+’ in Panel D). The lack of biotinylation (no NTPDase3 is detected in lanes ‘B’, Panel C) implies that little or no NTPDase3 immobilized on plates (marked with ‘0’ in Panel D). ( E ) Qualitative estimation of the ratio of the amount of hN3-B3 S antibody bound to plates (chemiluminescence intensity, Panel B) to the amount of NTPDase3 immobilized on plates (Panel D). Values above zero, zero and undefined are denoted by ‘+’, ‘0’ and ‘0/0’, respectively. ( F ) Antibody binding to immobilized NTPDase3 (denoted by ‘+’, Panel E) suggests epitope accessibility (denoted by ‘a’ in Panel F). The lack of antibody binding to immobilized enzyme (marked with ‘0’ in Panel E) suggests that the epitope is masked (marked with ‘M’ in Panel F). The 0/0 ratio for mutants A267C and D414C (Panel E) makes the evaluation of epitope accessibility uncertain.

    Article Snippet: Biotinylated membrane preparations (60 µg) were solubilized with 1% Triton X-100 containing 1 mM EDTA and 1% of Protease Inhibitor Cocktail Set III (Calbiochem) in a total volume of approximately 100 µl for 15 min at 21°C, and then centrifuged in an Airfuge for 20 min at 100 000× g .

    Techniques: Binding Assay, Incubation, Mutagenesis, Software, Transfection, Plasmid Preparation, Western Blot

    Site-directed tethering of biotinylated NTPDase3 to streptavidin-coated plates, and the effect of hN3-B3s inhibitory antibody on ATPase activity of tethered enzymes. ( A ) ATPase activity of biotinylated cysteine mutants. Crude membrane preparations were obtained from COS transfected with mutant NTPDase3 plasmid cDNA, as indicated below the bars, and biotinylated before harvesting. C10S NTPDase3 mutant was used as the background for all cysteine mutations. This mutant does not contain a free cysteine residue in the extracellular part of the protein and is shown for comparison (see ‘Materials and Methods’ for details). ATPase activities were assayed using the same amount of membrane protein, and the activities are expressed in µmole of inorganic phosphate (Pi) released per h and per mg of crude membrane protein. ( B ) ATPase activity of biotinylated NTPDase3 mutants immobilized on streptavidin-coated plates. Equal amount of biotinylated membrane preparation of each mutant was treated with Triton X-100, and the extracted proteins were immobilized on streptavidin-coated plates. ATPase activity is expressed in nmole of inorganic phosphate (Pi) released per h and per plate well. ( C ) Effect of hN3-B3s inhibitory antibody on ATPase activity of immobilized NTPDase3 mutants. Biotinylated NTPDase3 mutants immobilized on streptavidin-coated plates were incubated with either hN3-B3 S , or a BSA solution with no antibody (control), followed by ATPase assay. Values represent means ± SD for three or four plate wells. LA, enzymatic activity is too low to measure accurately. NA, no ATPase activity above the C10S background was detected.

    Journal: Protein Engineering, Design and Selection

    Article Title: Epitope mapping in cell surface proteins by site-directed masking: defining the structural elements of NTPDase3 inhibition by a monoclonal antibody

    doi: 10.1093/protein/gzq027

    Figure Lengend Snippet: Site-directed tethering of biotinylated NTPDase3 to streptavidin-coated plates, and the effect of hN3-B3s inhibitory antibody on ATPase activity of tethered enzymes. ( A ) ATPase activity of biotinylated cysteine mutants. Crude membrane preparations were obtained from COS transfected with mutant NTPDase3 plasmid cDNA, as indicated below the bars, and biotinylated before harvesting. C10S NTPDase3 mutant was used as the background for all cysteine mutations. This mutant does not contain a free cysteine residue in the extracellular part of the protein and is shown for comparison (see ‘Materials and Methods’ for details). ATPase activities were assayed using the same amount of membrane protein, and the activities are expressed in µmole of inorganic phosphate (Pi) released per h and per mg of crude membrane protein. ( B ) ATPase activity of biotinylated NTPDase3 mutants immobilized on streptavidin-coated plates. Equal amount of biotinylated membrane preparation of each mutant was treated with Triton X-100, and the extracted proteins were immobilized on streptavidin-coated plates. ATPase activity is expressed in nmole of inorganic phosphate (Pi) released per h and per plate well. ( C ) Effect of hN3-B3s inhibitory antibody on ATPase activity of immobilized NTPDase3 mutants. Biotinylated NTPDase3 mutants immobilized on streptavidin-coated plates were incubated with either hN3-B3 S , or a BSA solution with no antibody (control), followed by ATPase assay. Values represent means ± SD for three or four plate wells. LA, enzymatic activity is too low to measure accurately. NA, no ATPase activity above the C10S background was detected.

    Article Snippet: Biotinylated membrane preparations (60 µg) were solubilized with 1% Triton X-100 containing 1 mM EDTA and 1% of Protease Inhibitor Cocktail Set III (Calbiochem) in a total volume of approximately 100 µl for 15 min at 21°C, and then centrifuged in an Airfuge for 20 min at 100 000× g .

    Techniques: Activity Assay, Transfection, Mutagenesis, Plasmid Preparation, Incubation, ATPase Assay