tris buffered saline tbs  (Thermo Fisher)


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  • 99
    Name:
    TBS Buffer
    Description:
    Tris Buffered Saline TBS Buffer is intended for use with PanPath products TBS is supplied as a lyophilized powder and when dissolved in one liter distilled or deionised water it becomes 0 05M TBS pH 8 0 0 238M NaCl 0 0027M KCl with 0 05 Tween 20
    Catalog Number:
    r017r.0000
    Price:
    None
    Applications:
    Cell Analysis|Cellular Imaging|Chromogenic In Situ Hybridization|In Situ Hybridization (ISH)
    Category:
    Lab Reagents and Chemicals
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    Structured Review

    Thermo Fisher tris buffered saline tbs
    Tris Buffered Saline TBS Buffer is intended for use with PanPath products TBS is supplied as a lyophilized powder and when dissolved in one liter distilled or deionised water it becomes 0 05M TBS pH 8 0 0 238M NaCl 0 0027M KCl with 0 05 Tween 20
    https://www.bioz.com/result/tris buffered saline tbs/product/Thermo Fisher
    Average 99 stars, based on 251 article reviews
    Price from $9.99 to $1999.99
    tris buffered saline tbs - by Bioz Stars, 2020-11
    99/100 stars

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    Incubation:

    Article Title: Epitope mapping in cell surface proteins by site-directed masking: defining the structural elements of NTPDase3 inhibition by a monoclonal antibody
    Article Snippet: .. The supernatants were diluted with 1.2 ml TBS buffer (150 mM NaCl, 50 mM Tris–HCl, pH 7.5) containing 1% Triton X-100, and incubated with 15 µl ImmunoPure Immobilized Streptavidin agarose beads (Pierce) for 20 h with end-over-end rotation at 4°C. .. Beads were then washed three times with TBS containing 1% Triton X-100, and biotinylated proteins were eluted by re-suspending the beads in SDS loading buffer containing 200 mM DTT and boiling for 5 min. Beads were removed by centrifugation, and the supernatant was analyzed by western blotting for detection of biotinylated NTPDase3.

    Article Title: Binding Behavior of Microbial Functional Amyloids on Solid Surfaces
    Article Snippet: .. The blot was incubated in 5% non-fat dried milk in tris-buffered saline, 0.1% Tween 20 (TBS-T) at room temperature for an hour in order to block non-specific binding sites, then probed with 1:5,000 dilution of anti-his mouse primary antibody (Thermo Scientific Pierce) in TBS-T. After washing twice with TBS-T, the blot probed with 1:5,000 dilution of anti-mouse horseradish-peroxidase (HRP) conjugated secondary antibody (Thermo Scientific Pierce) in TBS-T. .. The membrane blot was rewashed with TBS-T and visualized with the Chemidoc MP imaging system (Bio-Rad) following an enhanced chemiluminescence (ECL) substrate (Bio-Rad) application.

    Article Title: Delta-secretase-cleaved Tau antagonizes TrkB neurotrophic signalings, mediating Alzheimer’s disease pathologies
    Article Snippet: .. After being washed with TBS solution, the sections were incubated with a mixture of Alexa Fluor 488- and 568-coupled secondary antibodies (Invitrogen) for detection. .. Images were acquired with confocal imaging (FV1000; Olympus).

    Article Title: Reduced inflammation and cytokine production in NKLAM deficient mice during Streptococcus pneumoniae infection
    Article Snippet: .. After washing in Tris-buffered saline (TBS) the sections were incubated with anti-rabbit HRP for 60 min followed by amplification with alexafluor-594 tyramide (Invitrogen). .. For the myeloperoxidase (MPO) staining, lung sections were treated with 0.3% H2 O2 , blocked and incubated with 1:100 anti-MPO (Abcam #ab9535).

    Article Title: The Aβ protofibril selective antibody mAb158 prevents accumulation of Aβ in astrocytes and rescues neurons from Aβ-induced cell death
    Article Snippet: .. The membrane was briefly washed in Tris-buffered saline (TBS) and blocked in 5% nonfat dry milk in 0.1% TBS-Tween for 1 h at RT, before the primary antibody (polyclonal rabbit anti-Aβ42 , 1:2000, Invitrogen) was added and incubated O/N at RT on shaking. .. The membrane was washed in 0.1% TBS-Tween for 2 × 5 min and 2 × 10 min prior to 1 h incubation with secondary anti-rabbit antibody conjugated with horseradish peroxidase (Pierce) in 0.1% TBS-Tween.

    Amplification:

    Article Title: Reduced inflammation and cytokine production in NKLAM deficient mice during Streptococcus pneumoniae infection
    Article Snippet: .. After washing in Tris-buffered saline (TBS) the sections were incubated with anti-rabbit HRP for 60 min followed by amplification with alexafluor-594 tyramide (Invitrogen). .. For the myeloperoxidase (MPO) staining, lung sections were treated with 0.3% H2 O2 , blocked and incubated with 1:100 anti-MPO (Abcam #ab9535).

    Binding Assay:

    Article Title: Binding Behavior of Microbial Functional Amyloids on Solid Surfaces
    Article Snippet: .. The blot was incubated in 5% non-fat dried milk in tris-buffered saline, 0.1% Tween 20 (TBS-T) at room temperature for an hour in order to block non-specific binding sites, then probed with 1:5,000 dilution of anti-his mouse primary antibody (Thermo Scientific Pierce) in TBS-T. After washing twice with TBS-T, the blot probed with 1:5,000 dilution of anti-mouse horseradish-peroxidase (HRP) conjugated secondary antibody (Thermo Scientific Pierce) in TBS-T. .. The membrane blot was rewashed with TBS-T and visualized with the Chemidoc MP imaging system (Bio-Rad) following an enhanced chemiluminescence (ECL) substrate (Bio-Rad) application.

    Blocking Assay:

    Article Title: Binding Behavior of Microbial Functional Amyloids on Solid Surfaces
    Article Snippet: .. The blot was incubated in 5% non-fat dried milk in tris-buffered saline, 0.1% Tween 20 (TBS-T) at room temperature for an hour in order to block non-specific binding sites, then probed with 1:5,000 dilution of anti-his mouse primary antibody (Thermo Scientific Pierce) in TBS-T. After washing twice with TBS-T, the blot probed with 1:5,000 dilution of anti-mouse horseradish-peroxidase (HRP) conjugated secondary antibody (Thermo Scientific Pierce) in TBS-T. .. The membrane blot was rewashed with TBS-T and visualized with the Chemidoc MP imaging system (Bio-Rad) following an enhanced chemiluminescence (ECL) substrate (Bio-Rad) application.

    Software:

    Article Title: Uptake of the antifungal cationic peptide Histatin 5 by Candida albicans Ssa2p requires binding to non-conventional sites within the ATPase domain
    Article Snippet: .. After washing six times with TBS buffer, membranes were developed with SuperSignal West Pico Chemiluminescent Substrate (150 μl cm−2 ) (Pierce) for 5 min and scanned using a Fuji LAS-1000 Plus Chemiluminescence Imager, and signals were quantified using ImageJ software (NIH). .. Repeatability of binding signals was confirmed by reprobing each membrane following regeneration with regeneration buffer (62.5 mM Tris-HCl, 2% SDS, 0.7% 2-mercaptoethanol, pH 6.7) for 30 min at 50°C.

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  • 95
    Thermo Fisher rabbit dylight 550 secondary antibody
    Allicin reduces the expression/activity of CD68 and MPO. (a) Confocal microscopic analysis of CD68 in the control and experimental animals. The experimental details are described in Materials and Methods. The secondary antibody used in this study was tagged with DyLight 550, and the cells were counterstained with Hoechst. The control animals exhibited lower CD68 expression. DSS treatment increased the expression of CD68 (Dylight 550; red). However, allicin treatment decreased the expression of CD68. (b) Activity of MPO. The MPO activity is presented as mU/mg of protein. The values are expressed as the means ± S.D. In comparison of   a control versus DSS,  b DSS versus DSS + allicin. “∗” denotes a statistically significant difference at  P
    Rabbit Dylight 550 Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit dylight 550 secondary antibody/product/Thermo Fisher
    Average 95 stars, based on 18 article reviews
    Price from $9.99 to $1999.99
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    90
    Thermo Fisher anti fibromodulin
    Identification of <t>fibromodulin</t> binding sites on collagen. A and B , collagen Toolkit II ( A ) and III ( B ) peptides were coated on a 96-well plate. The plate was blocked with BSA and incubated with biotinylated FMOD at 10 μg/ml (170 n m ) for 2 h. Binding was detected with streptavidin-HRP followed by TMB substrate, quenching with sulfuric acid, and measuring A 450 . BSA was used as a negative control. C , inhibition of FMOD binding to coated collagen I. The assay was performed as above, but collagen I was coated on a 96-well plate, and FMOD was preincubated with increasing amounts of the interacting Toolkit peptides III-5, III-44, and III-53 or with a negative control peptide, II-26. D , sequences of the interacting Toolkit peptides are listed, with homologue amino acids underlined (except the repetitive glycines). The non-binding peptides are shown in italics. E , homology alignment of FMOD-binding Toolkit peptides and the corresponding sequences on collagen I α1 and α2 chains. F , solid-phase binding assay to assess interaction of FMOD with cross-linking site-containing collagen peptides and their mutated variants. The assay was performed as in A . Peptide sequences are listed on the x axis ( K(Ac) , acetylated lysine), except the III-5 sequence, which is listed in E. These shorter peptides were synthesized with GCP terminal triplets. All error bars represent mean ± S.D. ( n = 3 technical replicates). All experiments were performed three times.
    Anti Fibromodulin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 90 stars, based on 1 article reviews
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    85
    Thermo Fisher gst tb nsmase membranes
    Expression, purification and activity of recombinant Tb <t>nSMase.</t> A. Tb nSMase was cloned and expressed as an N-terminal <t>GST</t> fusion protein in C43 E. coli . Protein samples after cell disruption were separated on a 10% SDS-PAGE gel, transferred to membrane and detected with anti-GST antibody. Lane 1, soluble protein (supernatant from 100 000 g ); lane 2, membrane fraction (pellet from 100 000 g ); lane 3, insoluble fraction (pellet from 45 000 g ). B. The substrate [ 3 H]-sphingomyelin (SM) and the product [ 3 H]-choline–phosphate of the Tb nSMase assay were separated by phase partitioning (organic and aqueous respectively), analysed by HPTLC and detected by autoradiography as described in Experimental procedures . Lane 1, negative control, [ 3 H]-SM with no membranes; lane 2, [ 3 H]-SM with Tb nSMase; lane 3, [ 3 H]-SM with Tb nSMase in presence of EDTA (5 mM); lane 4, negative control, [ 3 H]-SM with non expressing Tb nSMase membranes; lane 5, [ 3 H]-SM with non-expressing Tb nSMase membranes and EDTA (5 mM).; lane 6, [ 3 H]-PC with no membranes; lane 7, [ 3 H]-PC with Tb nSMase; lane 8, [ 3 H]-PC with Tb nSMase in the presence of EDTA (5 mM). Percentages of substrate turnover are shown, as determined by densitometry (ImageJ software). C. Tb nSMase activity was determined by end-point radioactive assay as a function of pH, using with MES buffer (filled triangles) or Bis-Tris Propane (filled squares) as described in Experimental procedures.
    Gst Tb Nsmase Membranes, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 85 stars, based on 1 article reviews
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    Allicin reduces the expression/activity of CD68 and MPO. (a) Confocal microscopic analysis of CD68 in the control and experimental animals. The experimental details are described in Materials and Methods. The secondary antibody used in this study was tagged with DyLight 550, and the cells were counterstained with Hoechst. The control animals exhibited lower CD68 expression. DSS treatment increased the expression of CD68 (Dylight 550; red). However, allicin treatment decreased the expression of CD68. (b) Activity of MPO. The MPO activity is presented as mU/mg of protein. The values are expressed as the means ± S.D. In comparison of   a control versus DSS,  b DSS versus DSS + allicin. “∗” denotes a statistically significant difference at  P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Allicin Alleviates Dextran Sodium Sulfate- (DSS-) Induced Ulcerative Colitis in BALB/c Mice

    doi: 10.1155/2015/605208

    Figure Lengend Snippet: Allicin reduces the expression/activity of CD68 and MPO. (a) Confocal microscopic analysis of CD68 in the control and experimental animals. The experimental details are described in Materials and Methods. The secondary antibody used in this study was tagged with DyLight 550, and the cells were counterstained with Hoechst. The control animals exhibited lower CD68 expression. DSS treatment increased the expression of CD68 (Dylight 550; red). However, allicin treatment decreased the expression of CD68. (b) Activity of MPO. The MPO activity is presented as mU/mg of protein. The values are expressed as the means ± S.D. In comparison of   a control versus DSS, b DSS versus DSS + allicin. “∗” denotes a statistically significant difference at P

    Article Snippet: After the sections were washed three times with TBS, the slides were incubated with goat and rabbit DyLight 550 secondary antibody (Thermo Scientific, Rockford, IL, USA) diluted 1 : 200 with TBS and incubated in the dark for 120 min at room temperature.

    Techniques: Expressing, Activity Assay

    Identification of fibromodulin binding sites on collagen. A and B , collagen Toolkit II ( A ) and III ( B ) peptides were coated on a 96-well plate. The plate was blocked with BSA and incubated with biotinylated FMOD at 10 μg/ml (170 n m ) for 2 h. Binding was detected with streptavidin-HRP followed by TMB substrate, quenching with sulfuric acid, and measuring A 450 . BSA was used as a negative control. C , inhibition of FMOD binding to coated collagen I. The assay was performed as above, but collagen I was coated on a 96-well plate, and FMOD was preincubated with increasing amounts of the interacting Toolkit peptides III-5, III-44, and III-53 or with a negative control peptide, II-26. D , sequences of the interacting Toolkit peptides are listed, with homologue amino acids underlined (except the repetitive glycines). The non-binding peptides are shown in italics. E , homology alignment of FMOD-binding Toolkit peptides and the corresponding sequences on collagen I α1 and α2 chains. F , solid-phase binding assay to assess interaction of FMOD with cross-linking site-containing collagen peptides and their mutated variants. The assay was performed as in A . Peptide sequences are listed on the x axis ( K(Ac) , acetylated lysine), except the III-5 sequence, which is listed in E. These shorter peptides were synthesized with GCP terminal triplets. All error bars represent mean ± S.D. ( n = 3 technical replicates). All experiments were performed three times.

    Journal: The Journal of Biological Chemistry

    Article Title: Fibromodulin Interacts with Collagen Cross-linking Sites and Activates Lysyl Oxidase *

    doi: 10.1074/jbc.M115.693408

    Figure Lengend Snippet: Identification of fibromodulin binding sites on collagen. A and B , collagen Toolkit II ( A ) and III ( B ) peptides were coated on a 96-well plate. The plate was blocked with BSA and incubated with biotinylated FMOD at 10 μg/ml (170 n m ) for 2 h. Binding was detected with streptavidin-HRP followed by TMB substrate, quenching with sulfuric acid, and measuring A 450 . BSA was used as a negative control. C , inhibition of FMOD binding to coated collagen I. The assay was performed as above, but collagen I was coated on a 96-well plate, and FMOD was preincubated with increasing amounts of the interacting Toolkit peptides III-5, III-44, and III-53 or with a negative control peptide, II-26. D , sequences of the interacting Toolkit peptides are listed, with homologue amino acids underlined (except the repetitive glycines). The non-binding peptides are shown in italics. E , homology alignment of FMOD-binding Toolkit peptides and the corresponding sequences on collagen I α1 and α2 chains. F , solid-phase binding assay to assess interaction of FMOD with cross-linking site-containing collagen peptides and their mutated variants. The assay was performed as in A . Peptide sequences are listed on the x axis ( K(Ac) , acetylated lysine), except the III-5 sequence, which is listed in E. These shorter peptides were synthesized with GCP terminal triplets. All error bars represent mean ± S.D. ( n = 3 technical replicates). All experiments were performed three times.

    Article Snippet: Antigen retrieval solution (L.A.B. solution from Polysciences) was used before quenching peroxidase with 0.3% hydrogen peroxide for 15 min and blocking the sections with 10% goat serum in TBS for 1 h. Slides were incubated with anti-fibromodulin (2 μg/ml in TBS and 1% serum) for 2 h, washed with TBS, and stained with the ultrasensitive ABC rabbit IgG staining kit (Thermo Scientific) and diaminobenzidine peroxidase substrate kit (Vector Labs).

    Techniques: Binding Assay, Incubation, Negative Control, Inhibition, Sequencing, Synthesized

    Proposed model for collagen cross-linking modulation by fibromodulin. A , alignment of the collagen III monomers is derived from 234-residue D periods. The identified high-affinity FMOD-binding sites in collagen are marked in dark green (III-5), yellow (III-8), blue (III-44), and light green (III-53). D periods are delimited with black lines . Collagen monomers are arranged in quarter-stagger, which reveals vertical alignment of III-44 and III-5 sites on neighboring collagen monomers. The two telopeptides ( red ) also align with FMOD-binding sites. B , proposed binding of the FMOD-LOX complex to a microfibril. FMOD binds across three monomers, interacting with the vertically aligned collagen-binding sites. LOX binds to the N-terminal end of FMOD and is positioned against the N-telopeptide in the microfibril.

    Journal: The Journal of Biological Chemistry

    Article Title: Fibromodulin Interacts with Collagen Cross-linking Sites and Activates Lysyl Oxidase *

    doi: 10.1074/jbc.M115.693408

    Figure Lengend Snippet: Proposed model for collagen cross-linking modulation by fibromodulin. A , alignment of the collagen III monomers is derived from 234-residue D periods. The identified high-affinity FMOD-binding sites in collagen are marked in dark green (III-5), yellow (III-8), blue (III-44), and light green (III-53). D periods are delimited with black lines . Collagen monomers are arranged in quarter-stagger, which reveals vertical alignment of III-44 and III-5 sites on neighboring collagen monomers. The two telopeptides ( red ) also align with FMOD-binding sites. B , proposed binding of the FMOD-LOX complex to a microfibril. FMOD binds across three monomers, interacting with the vertically aligned collagen-binding sites. LOX binds to the N-terminal end of FMOD and is positioned against the N-telopeptide in the microfibril.

    Article Snippet: Antigen retrieval solution (L.A.B. solution from Polysciences) was used before quenching peroxidase with 0.3% hydrogen peroxide for 15 min and blocking the sections with 10% goat serum in TBS for 1 h. Slides were incubated with anti-fibromodulin (2 μg/ml in TBS and 1% serum) for 2 h, washed with TBS, and stained with the ultrasensitive ABC rabbit IgG staining kit (Thermo Scientific) and diaminobenzidine peroxidase substrate kit (Vector Labs).

    Techniques: Derivative Assay, Binding Assay

    Lysyl oxidase activity in the presence of fibromodulin. Lysyl oxidase activity was measured with a commercial kit. As a sample, conditioned ( cond ) medium from 293T cells expressing LOX (or from LOX-negative control cells) was used. The medium was preincubated for 1 h with FMOD or its variants and then mixed with the substrate and incubated for 15 min at 37 °C. Enzyme activity was measured by fluorescence emission at 590 nm after excitation at 530 nm. The plotted values are biological duplicates. Similar results were obtained in three independent experiments.

    Journal: The Journal of Biological Chemistry

    Article Title: Fibromodulin Interacts with Collagen Cross-linking Sites and Activates Lysyl Oxidase *

    doi: 10.1074/jbc.M115.693408

    Figure Lengend Snippet: Lysyl oxidase activity in the presence of fibromodulin. Lysyl oxidase activity was measured with a commercial kit. As a sample, conditioned ( cond ) medium from 293T cells expressing LOX (or from LOX-negative control cells) was used. The medium was preincubated for 1 h with FMOD or its variants and then mixed with the substrate and incubated for 15 min at 37 °C. Enzyme activity was measured by fluorescence emission at 590 nm after excitation at 530 nm. The plotted values are biological duplicates. Similar results were obtained in three independent experiments.

    Article Snippet: Antigen retrieval solution (L.A.B. solution from Polysciences) was used before quenching peroxidase with 0.3% hydrogen peroxide for 15 min and blocking the sections with 10% goat serum in TBS for 1 h. Slides were incubated with anti-fibromodulin (2 μg/ml in TBS and 1% serum) for 2 h, washed with TBS, and stained with the ultrasensitive ABC rabbit IgG staining kit (Thermo Scientific) and diaminobenzidine peroxidase substrate kit (Vector Labs).

    Techniques: Activity Assay, Expressing, Negative Control, Incubation, Fluorescence

    Expression, purification and activity of recombinant Tb nSMase. A. Tb nSMase was cloned and expressed as an N-terminal GST fusion protein in C43 E. coli . Protein samples after cell disruption were separated on a 10% SDS-PAGE gel, transferred to membrane and detected with anti-GST antibody. Lane 1, soluble protein (supernatant from 100 000 g ); lane 2, membrane fraction (pellet from 100 000 g ); lane 3, insoluble fraction (pellet from 45 000 g ). B. The substrate [ 3 H]-sphingomyelin (SM) and the product [ 3 H]-choline–phosphate of the Tb nSMase assay were separated by phase partitioning (organic and aqueous respectively), analysed by HPTLC and detected by autoradiography as described in Experimental procedures . Lane 1, negative control, [ 3 H]-SM with no membranes; lane 2, [ 3 H]-SM with Tb nSMase; lane 3, [ 3 H]-SM with Tb nSMase in presence of EDTA (5 mM); lane 4, negative control, [ 3 H]-SM with non expressing Tb nSMase membranes; lane 5, [ 3 H]-SM with non-expressing Tb nSMase membranes and EDTA (5 mM).; lane 6, [ 3 H]-PC with no membranes; lane 7, [ 3 H]-PC with Tb nSMase; lane 8, [ 3 H]-PC with Tb nSMase in the presence of EDTA (5 mM). Percentages of substrate turnover are shown, as determined by densitometry (ImageJ software). C. Tb nSMase activity was determined by end-point radioactive assay as a function of pH, using with MES buffer (filled triangles) or Bis-Tris Propane (filled squares) as described in Experimental procedures.

    Journal: Molecular Microbiology

    Article Title: The essential neutral sphingomyelinase is involved in the trafficking of the variant surface glycoprotein in the bloodstream form of Trypanosoma brucei

    doi: 10.1111/j.1365-2958.2010.07151.x

    Figure Lengend Snippet: Expression, purification and activity of recombinant Tb nSMase. A. Tb nSMase was cloned and expressed as an N-terminal GST fusion protein in C43 E. coli . Protein samples after cell disruption were separated on a 10% SDS-PAGE gel, transferred to membrane and detected with anti-GST antibody. Lane 1, soluble protein (supernatant from 100 000 g ); lane 2, membrane fraction (pellet from 100 000 g ); lane 3, insoluble fraction (pellet from 45 000 g ). B. The substrate [ 3 H]-sphingomyelin (SM) and the product [ 3 H]-choline–phosphate of the Tb nSMase assay were separated by phase partitioning (organic and aqueous respectively), analysed by HPTLC and detected by autoradiography as described in Experimental procedures . Lane 1, negative control, [ 3 H]-SM with no membranes; lane 2, [ 3 H]-SM with Tb nSMase; lane 3, [ 3 H]-SM with Tb nSMase in presence of EDTA (5 mM); lane 4, negative control, [ 3 H]-SM with non expressing Tb nSMase membranes; lane 5, [ 3 H]-SM with non-expressing Tb nSMase membranes and EDTA (5 mM).; lane 6, [ 3 H]-PC with no membranes; lane 7, [ 3 H]-PC with Tb nSMase; lane 8, [ 3 H]-PC with Tb nSMase in the presence of EDTA (5 mM). Percentages of substrate turnover are shown, as determined by densitometry (ImageJ software). C. Tb nSMase activity was determined by end-point radioactive assay as a function of pH, using with MES buffer (filled triangles) or Bis-Tris Propane (filled squares) as described in Experimental procedures.

    Article Snippet: Protein was extracted from the GST–Tb nSMase membranes by solubilizing in 1% Triton X-100 prior to determining the total protein content using the BCA protein assay kit (Thermo Scientific).

    Techniques: Expressing, Purification, Activity Assay, Recombinant, Clone Assay, SDS Page, High Performance Thin Layer Chromatography, Autoradiography, Negative Control, Software, Radioactivity

    Activity of recombinant Tb nSMase. A. Reaction catalysed by Tb nSMase along with coupled the coupled Amplex red assay. AP, alkaline phosphatase; ChOx, choline oxidase; Cho-P, choline–phosphate; HRP, horseradish peroxidase. B. Determination of Tb nSMase Michaelis-Menten constants for SM (inserts show Lineweaver–Burk plot). C. Structure of manumycin A, a commercially available nSMase inhibitor. D. Enzyme activity of Tb nSMase in either washed bloodstream T. brucei membranes (lanes 1–3) or E. coli membranes expressing GST– Tb nSMase (lanes 4–6), after pre-incubation with either nothing (lanes 1 and 4) or miltefosine (lanes 2 and 5) or edelfosine (lanes 3 and 6) in the presence of SM as substrate as described in Experimental procedures . Insert shows structure of edelfosine.

    Journal: Molecular Microbiology

    Article Title: The essential neutral sphingomyelinase is involved in the trafficking of the variant surface glycoprotein in the bloodstream form of Trypanosoma brucei

    doi: 10.1111/j.1365-2958.2010.07151.x

    Figure Lengend Snippet: Activity of recombinant Tb nSMase. A. Reaction catalysed by Tb nSMase along with coupled the coupled Amplex red assay. AP, alkaline phosphatase; ChOx, choline oxidase; Cho-P, choline–phosphate; HRP, horseradish peroxidase. B. Determination of Tb nSMase Michaelis-Menten constants for SM (inserts show Lineweaver–Burk plot). C. Structure of manumycin A, a commercially available nSMase inhibitor. D. Enzyme activity of Tb nSMase in either washed bloodstream T. brucei membranes (lanes 1–3) or E. coli membranes expressing GST– Tb nSMase (lanes 4–6), after pre-incubation with either nothing (lanes 1 and 4) or miltefosine (lanes 2 and 5) or edelfosine (lanes 3 and 6) in the presence of SM as substrate as described in Experimental procedures . Insert shows structure of edelfosine.

    Article Snippet: Protein was extracted from the GST–Tb nSMase membranes by solubilizing in 1% Triton X-100 prior to determining the total protein content using the BCA protein assay kit (Thermo Scientific).

    Techniques: Activity Assay, Recombinant, Amplex Red Assay, Expressing, Incubation