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    Structured Review

    Millipore tris buffered saline
    ( A ) Fibrinolytic effects of rSKs on human plasma clots. Human plasma (45 μl) was mixed with trace amounts of 125 ). After a wash with <t>Tris-buffered</t> saline (pH 7.4) containing the thrombin inhibitor <t>PPACK</t> (10 μM), the clots were suspended in 2 ml of plasma containing 10 μM PPACK. Various amounts of rSK and rSKΔ59 that had undergone active-site titration (0–50 nM) were added to the plasma, and, after 6 h, the amount of fibrinolysis was determined by measuring the release of soluble 125 ). The means ± SD are shown (but in some cases the SD are too small to be visible). ( B ) Fibrinogen degradation by rSK activator complexes. At the end of the fibrinolysis experiments presented in A ). The means ± SD are shown (but in some cases the SD are too small to be visible).
    Tris Buffered Saline, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1958 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "A catalytic switch and the conversion of streptokinase to a fibrin-targeted plasminogen activator"

    Article Title: A catalytic switch and the conversion of streptokinase to a fibrin-targeted plasminogen activator

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    ( A ) Fibrinolytic effects of rSKs on human plasma clots. Human plasma (45 μl) was mixed with trace amounts of 125 ). After a wash with Tris-buffered saline (pH 7.4) containing the thrombin inhibitor PPACK (10 μM), the clots were suspended in 2 ml of plasma containing 10 μM PPACK. Various amounts of rSK and rSKΔ59 that had undergone active-site titration (0–50 nM) were added to the plasma, and, after 6 h, the amount of fibrinolysis was determined by measuring the release of soluble 125 ). The means ± SD are shown (but in some cases the SD are too small to be visible). ( B ) Fibrinogen degradation by rSK activator complexes. At the end of the fibrinolysis experiments presented in A ). The means ± SD are shown (but in some cases the SD are too small to be visible).
    Figure Legend Snippet: ( A ) Fibrinolytic effects of rSKs on human plasma clots. Human plasma (45 μl) was mixed with trace amounts of 125 ). After a wash with Tris-buffered saline (pH 7.4) containing the thrombin inhibitor PPACK (10 μM), the clots were suspended in 2 ml of plasma containing 10 μM PPACK. Various amounts of rSK and rSKΔ59 that had undergone active-site titration (0–50 nM) were added to the plasma, and, after 6 h, the amount of fibrinolysis was determined by measuring the release of soluble 125 ). The means ± SD are shown (but in some cases the SD are too small to be visible). ( B ) Fibrinogen degradation by rSK activator complexes. At the end of the fibrinolysis experiments presented in A ). The means ± SD are shown (but in some cases the SD are too small to be visible).

    Techniques Used: Titration

    2) Product Images from "ADAMTS-4_v1 Is a Splice Variant of ADAMTS-4 That Is Expressed as a Protein in Human Synovium and Cleaves Aggrecan at the Interglobular Domain"

    Article Title: ADAMTS-4_v1 Is a Splice Variant of ADAMTS-4 That Is Expressed as a Protein in Human Synovium and Cleaves Aggrecan at the Interglobular Domain

    Journal: Arthritis and Rheumatism

    doi: 10.1002/art.38102

    Characterization of recombinant ADAMTS-4_v1. A, Blot of anti–FLAG M2 monoclonal antibody (mAb) immunoprecipitates from Triton X-100 extracts of untransfected HEK 293 cells and ADAMTS4_v1 -pCEP4–transfected HEK 293 cells, following sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Coomassie brilliant blue R250 staining. The band marked with an asterisk yielded an N-terminal amino acid sequence of ASPLPRE…, whereas no sequence was obtained from the band marked with a double asterisk. B–D, Triplicate Western blots of anti–FLAG M2 mAb immunoprecipitates from Triton X-100 cell extracts and conditioned media obtained from untransfected, ADAMTS4.1 -pCEP4–transfected, or ADAMTS4_v1 -pCEP4–transfected HEK 293 cells, probed with anti–FLAG M2 mAb (B), a rabbit antiserum (ab28285) to the carboxyl-terminus of ADAMTS-4 (C), or a rabbit antiserum (ab39201) to the pro domain of ADAMTS-4 (D). E, Western blot of anti–FLAG M2 mAb immunoprecipitates from a Triton X-100 extract of an equal number of Tris buffered saline–washed ADAMTS4_v1 -pCEP4–transfected HEK 293 cells (Triton X-100 extract), a Triton X-100 extract of the pellet after high salt extraction (high salt pellet), and a 1 M NaCl, 50 m M Tris HCl, pH 7.4, extract of ADAMTS4_v1 -pCEP4–transfected HEK 293 cells (high salt extract). F, Western blot probed with anti–FLAG M2 mAb using a plasma membrane protein extraction kit to prepare cytosol, total membranes, and plasma membranes from ADAMTS4_v1 -pCEP4–transfected HEK 293 cells prior to anti–FLAG M2 mAb immunoprecipitation.
    Figure Legend Snippet: Characterization of recombinant ADAMTS-4_v1. A, Blot of anti–FLAG M2 monoclonal antibody (mAb) immunoprecipitates from Triton X-100 extracts of untransfected HEK 293 cells and ADAMTS4_v1 -pCEP4–transfected HEK 293 cells, following sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Coomassie brilliant blue R250 staining. The band marked with an asterisk yielded an N-terminal amino acid sequence of ASPLPRE…, whereas no sequence was obtained from the band marked with a double asterisk. B–D, Triplicate Western blots of anti–FLAG M2 mAb immunoprecipitates from Triton X-100 cell extracts and conditioned media obtained from untransfected, ADAMTS4.1 -pCEP4–transfected, or ADAMTS4_v1 -pCEP4–transfected HEK 293 cells, probed with anti–FLAG M2 mAb (B), a rabbit antiserum (ab28285) to the carboxyl-terminus of ADAMTS-4 (C), or a rabbit antiserum (ab39201) to the pro domain of ADAMTS-4 (D). E, Western blot of anti–FLAG M2 mAb immunoprecipitates from a Triton X-100 extract of an equal number of Tris buffered saline–washed ADAMTS4_v1 -pCEP4–transfected HEK 293 cells (Triton X-100 extract), a Triton X-100 extract of the pellet after high salt extraction (high salt pellet), and a 1 M NaCl, 50 m M Tris HCl, pH 7.4, extract of ADAMTS4_v1 -pCEP4–transfected HEK 293 cells (high salt extract). F, Western blot probed with anti–FLAG M2 mAb using a plasma membrane protein extraction kit to prepare cytosol, total membranes, and plasma membranes from ADAMTS4_v1 -pCEP4–transfected HEK 293 cells prior to anti–FLAG M2 mAb immunoprecipitation.

    Techniques Used: Recombinant, Transfection, Polyacrylamide Gel Electrophoresis, Staining, Sequencing, Western Blot, Protein Extraction, Immunoprecipitation

    3) Product Images from "Mesoporous silica nanoparticles trigger mitophagy in endothelial cells and perturb neuronal network activity in a size- and time-dependent manner"

    Article Title: Mesoporous silica nanoparticles trigger mitophagy in endothelial cells and perturb neuronal network activity in a size- and time-dependent manner

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S127663

    Analysis of MSNP protein coronas by SDS-PAGE. Notes: MSNPs of different sizes were incubated with cell-culture medium with 5% or 10% serum at 37°C for 24 hours. After centrifugation to remove unbound serum proteins, pellets were washed three times and then resuspended in 30 µL water and analyzed by SDS-PAGE on a 12% bis-Tris-glycine gel (Thermo Fisher Scientific), followed by EZ blue staining. Representative gels are shown. ( A ) Protein coronas of MSNPs after incubation in cell-culture medium with 5% serum. Lane 1, aliquot of medium; lane 2, protein coronas of 30 nm MSNPs; lane 3, protein coronas of 250 nm MSNPs; lane 4, medium alone submitted to centrifugation as negative control; lane 5, molecular weight marker. ( B ) Protein coronas of MSNPs after incubation in cell-culture medium with 10% serum. Lane 1, medium alone submitted to centrifugation as negative control; lane 2, protein coronas of 30 nm MSNPs; lane 3, protein coronas of 250 nm MSNPs; lane 4, an aliquot of medium; lane 5, molecular weight marker. Abbreviations: MSNP, mesoporous silica nanoparticle; SDS-PAGE, sodium dodecyl sulfate polyacrylamide-gel electrophoresis.
    Figure Legend Snippet: Analysis of MSNP protein coronas by SDS-PAGE. Notes: MSNPs of different sizes were incubated with cell-culture medium with 5% or 10% serum at 37°C for 24 hours. After centrifugation to remove unbound serum proteins, pellets were washed three times and then resuspended in 30 µL water and analyzed by SDS-PAGE on a 12% bis-Tris-glycine gel (Thermo Fisher Scientific), followed by EZ blue staining. Representative gels are shown. ( A ) Protein coronas of MSNPs after incubation in cell-culture medium with 5% serum. Lane 1, aliquot of medium; lane 2, protein coronas of 30 nm MSNPs; lane 3, protein coronas of 250 nm MSNPs; lane 4, medium alone submitted to centrifugation as negative control; lane 5, molecular weight marker. ( B ) Protein coronas of MSNPs after incubation in cell-culture medium with 10% serum. Lane 1, medium alone submitted to centrifugation as negative control; lane 2, protein coronas of 30 nm MSNPs; lane 3, protein coronas of 250 nm MSNPs; lane 4, an aliquot of medium; lane 5, molecular weight marker. Abbreviations: MSNP, mesoporous silica nanoparticle; SDS-PAGE, sodium dodecyl sulfate polyacrylamide-gel electrophoresis.

    Techniques Used: SDS Page, Incubation, Cell Culture, Centrifugation, Staining, Negative Control, Molecular Weight, Marker, Polyacrylamide Gel Electrophoresis

    4) Product Images from "TAPBPR isoforms exhibit altered association with MHC class I"

    Article Title: TAPBPR isoforms exhibit altered association with MHC class I

    Journal: Immunology

    doi: 10.1111/imm.12253

    Altered trafficking of TAPBPR β . (a) Cytofluorometric analysis for surface TAPBPR expression on HeLa cells (black line histogram) and HeLa transduced with TAPBPR α (grey-filled histogram) or TAPBPR β (black dotted line). Staining of non-transduced HeLa cells with an isotype control is included as a negative control (grey-dotted histogram). (b) Bar graph of mean fluorescence intensity (MFI) of surface TAPBPR on the full panel of cells expression the TAPBPR isoforms from two independent experiments (Error bars: ± SEM). (c) The anterograde transport rate of TAPBPR β was compared with TAPBPR α expressed in HeLa cells using pulse–chase analysis. HeLa cells expressing TAPBPR α and TAPBPR β were labelled with [ 35 S]methionine/cysteine for 30 min and chased for 0–6 hr as indicated. Following lysis in 1% digitonin Tris-buffered saline, TAPBPR was isolated from pre-cleared post-nuclear supernatants by immunoprecipitation using the mouse monoclonal antibody PeTe4. Following elution and denaturation the samples were treated with or without endoglycosidase H (Endo H). The signal intensity of the MHC class I heavy chain band was determined by densitometry and the amount of Endo-H-resistant MHC class I associated with TAPBPR at each time-point was plotted as a percentage of the Endo-H-resistant MHC class I associated with TAPBPR at the 0 time-point.
    Figure Legend Snippet: Altered trafficking of TAPBPR β . (a) Cytofluorometric analysis for surface TAPBPR expression on HeLa cells (black line histogram) and HeLa transduced with TAPBPR α (grey-filled histogram) or TAPBPR β (black dotted line). Staining of non-transduced HeLa cells with an isotype control is included as a negative control (grey-dotted histogram). (b) Bar graph of mean fluorescence intensity (MFI) of surface TAPBPR on the full panel of cells expression the TAPBPR isoforms from two independent experiments (Error bars: ± SEM). (c) The anterograde transport rate of TAPBPR β was compared with TAPBPR α expressed in HeLa cells using pulse–chase analysis. HeLa cells expressing TAPBPR α and TAPBPR β were labelled with [ 35 S]methionine/cysteine for 30 min and chased for 0–6 hr as indicated. Following lysis in 1% digitonin Tris-buffered saline, TAPBPR was isolated from pre-cleared post-nuclear supernatants by immunoprecipitation using the mouse monoclonal antibody PeTe4. Following elution and denaturation the samples were treated with or without endoglycosidase H (Endo H). The signal intensity of the MHC class I heavy chain band was determined by densitometry and the amount of Endo-H-resistant MHC class I associated with TAPBPR at each time-point was plotted as a percentage of the Endo-H-resistant MHC class I associated with TAPBPR at the 0 time-point.

    Techniques Used: Expressing, Transduction, Staining, Negative Control, Fluorescence, Pulse Chase, Lysis, Isolation, Immunoprecipitation

    Association of the TAPBPR isoforms with MHC class I. (a) TAPBPR was isolated by immunoprecipitation (using polyclonal antiserum R039) from the panel of HeLa cells stably transduced with the TAPBPR isoforms lysed in 1% Triton X-100 Tris-buffered saline. As a negative control non-transfected HeLa cells were included (−). Western blot analysis was performed for TAPBPR using mouse anti-TAPBPR, the MHC class I heavy chain using HC10 or calnexin on TAPBPR immunoprecipitated or lysates as indicated. (b) Cytofluorometric analysis for surface HLA-A68 expression from HeLa cell (black line histogram) and HeLa transduced with TAPBPR α (grey-filled histogram) or TAPBPR β (black dotted line). Staining of non-transduced HeLa with an isotype control is included as a negative control (grey-dotted histogram). (c) Bar graph of mean fluorescence intensity (MFI) of surface HLA-A68 on the full panel of cells expression the TAPBPR isoforms from two independent experiments (Error bars: ± SEM).
    Figure Legend Snippet: Association of the TAPBPR isoforms with MHC class I. (a) TAPBPR was isolated by immunoprecipitation (using polyclonal antiserum R039) from the panel of HeLa cells stably transduced with the TAPBPR isoforms lysed in 1% Triton X-100 Tris-buffered saline. As a negative control non-transfected HeLa cells were included (−). Western blot analysis was performed for TAPBPR using mouse anti-TAPBPR, the MHC class I heavy chain using HC10 or calnexin on TAPBPR immunoprecipitated or lysates as indicated. (b) Cytofluorometric analysis for surface HLA-A68 expression from HeLa cell (black line histogram) and HeLa transduced with TAPBPR α (grey-filled histogram) or TAPBPR β (black dotted line). Staining of non-transduced HeLa with an isotype control is included as a negative control (grey-dotted histogram). (c) Bar graph of mean fluorescence intensity (MFI) of surface HLA-A68 on the full panel of cells expression the TAPBPR isoforms from two independent experiments (Error bars: ± SEM).

    Techniques Used: Isolation, Immunoprecipitation, Stable Transfection, Transduction, Negative Control, Transfection, Western Blot, Expressing, Staining, Fluorescence

    5) Product Images from "Shielding of a Lipooligosaccharide IgM Epitope Allows Evasion of Neutrophil-Mediated Killing of an Invasive Strain of Nontypeable Haemophilus influenzae"

    Article Title: Shielding of a Lipooligosaccharide IgM Epitope Allows Evasion of Neutrophil-Mediated Killing of an Invasive Strain of Nontypeable Haemophilus influenzae

    Journal: mBio

    doi: 10.1128/mBio.01478-14

    Binding of IgM to HepIII-β1,2-Glc increases neutrophil-mediated killing. (A) Predicted LOS structure of NTHi strain R2866 (23; Elke Schweda, personal communication) with phase-variable LOS synthesis genes in gray. (B) R2866 LOS mutants were incubated for 15 min with 5% NHS at 37°C, and binding of IgM to the bacterial surface was determined by flow cytometry. (C) R2866LOS mutants were incubated with 3% NHS with or without neutrophils, and survival was determined after incubation at 37°C for 30 min. (D) LOS was isolated from R2866 LOS mutants, separated by Tris Tricine SDS-PAGE, and visualized by silver staining, or IgM binding was detected by Western blot analysis. (E) R2866Δ lic1/lgtF/lic2A was incubated for 15 min with 5% NHS that was preincubated with LOS isolated from R2866Δ lic1/lgtF/lic2A , R2866Δ lic1/galE , or R2866Δ lpsA/lgtF for 15 min at 37°C, and binding of IgM to the bacterial surface was determined by flow cytometry. Statistical significance was determined with a one-way analysis of variance and the Tukey post hoc test (B and C) or two-way analysis of variance and the Bonferroni post hoc test (E). **, P
    Figure Legend Snippet: Binding of IgM to HepIII-β1,2-Glc increases neutrophil-mediated killing. (A) Predicted LOS structure of NTHi strain R2866 (23; Elke Schweda, personal communication) with phase-variable LOS synthesis genes in gray. (B) R2866 LOS mutants were incubated for 15 min with 5% NHS at 37°C, and binding of IgM to the bacterial surface was determined by flow cytometry. (C) R2866LOS mutants were incubated with 3% NHS with or without neutrophils, and survival was determined after incubation at 37°C for 30 min. (D) LOS was isolated from R2866 LOS mutants, separated by Tris Tricine SDS-PAGE, and visualized by silver staining, or IgM binding was detected by Western blot analysis. (E) R2866Δ lic1/lgtF/lic2A was incubated for 15 min with 5% NHS that was preincubated with LOS isolated from R2866Δ lic1/lgtF/lic2A , R2866Δ lic1/galE , or R2866Δ lpsA/lgtF for 15 min at 37°C, and binding of IgM to the bacterial surface was determined by flow cytometry. Statistical significance was determined with a one-way analysis of variance and the Tukey post hoc test (B and C) or two-way analysis of variance and the Bonferroni post hoc test (E). **, P

    Techniques Used: Binding Assay, Gas Chromatography, Incubation, Flow Cytometry, Cytometry, Isolation, SDS Page, Silver Staining, Western Blot

    Binding of IgM is specific for an epitope that includes HepIII-β1,2-Glc, but not HepIII-β1,2-Gal. (A) Predicted LOS structures of RdΔ lic1/lgtF/lic2A and (B) RdΔ lic1/lgtF/lic2A with lpsA gene from R2846. (C) RdΔ lic1/lgtF/lic2A and RdΔ lic1/lgtF/lic2A R2846_ lpsA mutants were incubated for 15 min with 5% NHS at 37°C and binding of IgM to the bacterial surface was determined by flow cytometry. (D) LOS was isolated from RdΔ lic1/lgtF/lic2A and RdΔ lic1/lgtF/lic2A R2846_ lpsA mutants, separated by Tris-Tricine SDS-PAGE, and visualized by silver staining or IgM binding was detected by Western blotting. (H) RdΔ lic1/lgtF/lic2A and RdΔ lic1/lgtF/lic2A R2846_ lpsA mutants were incubated with 1% NHS with or without neutrophils, and survival was determined after incubation at 37°C for 30 min. Statistical significance was determined using an unpaired Student t test. **, P
    Figure Legend Snippet: Binding of IgM is specific for an epitope that includes HepIII-β1,2-Glc, but not HepIII-β1,2-Gal. (A) Predicted LOS structures of RdΔ lic1/lgtF/lic2A and (B) RdΔ lic1/lgtF/lic2A with lpsA gene from R2846. (C) RdΔ lic1/lgtF/lic2A and RdΔ lic1/lgtF/lic2A R2846_ lpsA mutants were incubated for 15 min with 5% NHS at 37°C and binding of IgM to the bacterial surface was determined by flow cytometry. (D) LOS was isolated from RdΔ lic1/lgtF/lic2A and RdΔ lic1/lgtF/lic2A R2846_ lpsA mutants, separated by Tris-Tricine SDS-PAGE, and visualized by silver staining or IgM binding was detected by Western blotting. (H) RdΔ lic1/lgtF/lic2A and RdΔ lic1/lgtF/lic2A R2846_ lpsA mutants were incubated with 1% NHS with or without neutrophils, and survival was determined after incubation at 37°C for 30 min. Statistical significance was determined using an unpaired Student t test. **, P

    Techniques Used: Binding Assay, Gas Chromatography, Incubation, Flow Cytometry, Cytometry, Isolation, SDS Page, Silver Staining, Western Blot

    Phase-variable incorporation of galactose on HepIII-β1,2-Glc blocks binding of IgM and neutrophil-mediated killing. (A) Predicted LOS structure of Rd ( 25 ) with phase-variable LOS synthesis genes in gray. (B) Rd LOS mutants were incubated for 15 min with 5% NHS at 37°C, and binding of IgM to the bacterial surface was determined by flow cytometry. (C) RdLOS mutants were incubated with 1% NHS with or without neutrophils, and survival was determined after incubation at 37°C for 30 min. (D) LOS was isolated from Rd LOS mutants, separated by Tris Tricine SDS-PAGE, and visualized by silver staining, or IgM binding was detected by Western blot analysis. (E) RdΔ lic1/lgtF mutants exposed to 1% NHS with neutrophils for three rounds were incubated for 15 min with 5% NHS at 37°C, and binding of IgM to the bacterial surface was determined by flow cytometry. (F) RdΔ lic1/lgtF mutants exposed to 1% NHS with neutrophils were incubated with 1% NHS with or without neutrophils for three rounds, and survival was determined after incubation at 37°C for 30 min. (G) RdΔ lic1/lgtF/lic2A mutants exposed to 1% NHS with neutrophils were incubated with 1% NHS with or without neutrophils for three rounds, and survival was determined after incubation at 37°C for 30 min. Statistical significance was determined with a one-way analysis of variance and the Tukey post hoc test. **, P
    Figure Legend Snippet: Phase-variable incorporation of galactose on HepIII-β1,2-Glc blocks binding of IgM and neutrophil-mediated killing. (A) Predicted LOS structure of Rd ( 25 ) with phase-variable LOS synthesis genes in gray. (B) Rd LOS mutants were incubated for 15 min with 5% NHS at 37°C, and binding of IgM to the bacterial surface was determined by flow cytometry. (C) RdLOS mutants were incubated with 1% NHS with or without neutrophils, and survival was determined after incubation at 37°C for 30 min. (D) LOS was isolated from Rd LOS mutants, separated by Tris Tricine SDS-PAGE, and visualized by silver staining, or IgM binding was detected by Western blot analysis. (E) RdΔ lic1/lgtF mutants exposed to 1% NHS with neutrophils for three rounds were incubated for 15 min with 5% NHS at 37°C, and binding of IgM to the bacterial surface was determined by flow cytometry. (F) RdΔ lic1/lgtF mutants exposed to 1% NHS with neutrophils were incubated with 1% NHS with or without neutrophils for three rounds, and survival was determined after incubation at 37°C for 30 min. (G) RdΔ lic1/lgtF/lic2A mutants exposed to 1% NHS with neutrophils were incubated with 1% NHS with or without neutrophils for three rounds, and survival was determined after incubation at 37°C for 30 min. Statistical significance was determined with a one-way analysis of variance and the Tukey post hoc test. **, P

    Techniques Used: Gas Chromatography, Binding Assay, Incubation, Flow Cytometry, Cytometry, Isolation, SDS Page, Silver Staining, Western Blot

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    Incubation:

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    Protease Inhibitor:

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    Transfection:

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    Labeling:

    Article Title: Effect of the Calpain System on Volatile Flavor Compounds in the BeefLongissimus lumborum Muscle
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    Immunoprecipitation:

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    other:

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    Fractionation:

    Article Title: Formation and spreading of TDP-43 aggregates in cultured neuronal and glial cells demonstrated by time-lapse imaging
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    Imaging:

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    Cell Fractionation:

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    Article Snippet: .. Cell fractionation For sarkosyl fractionation, cells were lysed and sonicated in Tris buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA and 5 mM EGTA) containing phosphatase inhibitors (5 mM NaF and 2 mM Na3 VO4 ) and 1× protease inhibitor cocktail (Millipore, Billerica, MA, USA), followed by centrifugation at 160,000 × g for 20 min at 4°C. ..

    Sonication:

    Article Title: Formation and spreading of TDP-43 aggregates in cultured neuronal and glial cells demonstrated by time-lapse imaging
    Article Snippet: .. Cell fractionation For sarkosyl fractionation, cells were lysed and sonicated in Tris buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA and 5 mM EGTA) containing phosphatase inhibitors (5 mM NaF and 2 mM Na3 VO4 ) and 1× protease inhibitor cocktail (Millipore, Billerica, MA, USA), followed by centrifugation at 160,000 × g for 20 min at 4°C. ..

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  • 99
    Millipore anti scn9a monoclonal antibody
    Isolation of TALE-VP16 fusions targeting the human <t>SCN9A</t> gene using the 11-mer TALE-VP16 library and the yeast one-hybrid assay. (a) Schematic illustration of the yeast one-hybrid assay using the 11-mer TALE-VP16 library. A bait sequence was cloned in front of an antibiotic resistance gene (Aba resistance gene) in yeast. The 11-mer TALE-VP16 library was then transformed into this stable clone and a surviving assay was performed on -Leu plates containing 100 nM Aba. (b) The RVD sequences of isolated TALE-VP16 fusions and their targets within the human SCN9A bait sequence (scale not proportional). TALE-VP16 fusions were shown to bind to both the plus and the minus strands of the SCN9A bait sequence. (c) The isolated TALE-VP16 fusions induced overexpression of endogenous SCN9A in HEK293 cells and A431 cells. The mRNA levels of SCN9A were determined by quantitative RT-PCR. An empty vector (PEF-1) was used as the control. Columns 1-6: All TALE-VP16 fusions were able to effectively induce the overexpression of SCN9A in HEK293 cells (n = 5). Columns 11-16: All TALE-VP16 fusions effectively induced the overexpression of SCN9A in A431 cells (n = 3). Columns 7–10: All 4 TALEs designed according to TALE-NT 2.0 failed to induce the overexpression of SCN9A in HEK293 cells (n = 3). Inlet: Western blot shows that all TALE-VP16 fusions induced the overexpression of SCN9A protein in A431 cells (representative data of two independent experiments). (d) The RVD sequences of isolated TALE-VP16 fusions and their targets within the human miR-34b/c bait sequence (scale not proportional). (e) Confirmation of the binding between isolated TALE-VP16 fusion M1 and its predicted gene target within the human miR-34b/c bait sequence. The isolated clone M1 was predicted to target 5′-TTTCTAGGTAT-3′ within the miR-34b/c bait sequence. The full-length bait sequence (pAbAi-miR-34b/c) or bait with the predicted target site deleted (pAbAi-miR-34b/c (ΔTTTCTAGGTAT)) was stably integrated into yeast cells. TALE-VP16 fusion clone M1 was then transformed into either cell line. Only cells which contained the intact bait sequence survived the 100 nM Aba selection. (f) The isolated TALE-VP16 fusion M1 effectively induced overexpression of miR-34b in a dose-dependent manner in both HEK293 and HeLa cells (n = 3 for both cell lines).
    Anti Scn9a Monoclonal Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 1 article reviews
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    anti scn9a monoclonal antibody - by Bioz Stars, 2020-08
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    91
    Millipore biotinylated chondroadherin
    Collagen binding by <t>chondroadherin.</t> (A) Binding of <t>biotinylated</t> chondroadherin to immobilised Collagen Toolkit II peptides. Binding was detected with streptavidin-HRP and TMB substrate (absorbance at 450 nm). Error bars show standard deviation from three technical replicates. (B) Dose-response curve of chondroadherin binding to peptide II-26 peptide interaction. (C) Inhibition of the chondroadherin-collagen II interaction by peptide II-26 peptide (solid line) or peptide III-8 (negative control, dashed line). (D) Binding of peptide AB31 to chondroadherin (CHAD) in solution analysed by size exclusion chromatography. AB31 and chondroadherin were mixed in a 2:1 molar ration and incubated for 15 min before injection onto the Superdex 75 column. The sequences of peptides II-26 and AB31 are indicated on the right and the sequence common to both is shaded.
    Biotinylated Chondroadherin, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/biotinylated chondroadherin/product/Millipore
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    99
    Millipore rnase a
    Polytene chromosome spreads of  D. melanogaster  wild type were treated with RNase A/RNase H mixture followed by proteinase K digestion in a time course experiment and subsequent immunological detection of triple-stranded DNA. DAPI staining (blue signal) and antibody labelling (red signal) were superimposed. Scale bar represents 25 µm.
    Rnase A, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 327 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Isolation of TALE-VP16 fusions targeting the human SCN9A gene using the 11-mer TALE-VP16 library and the yeast one-hybrid assay. (a) Schematic illustration of the yeast one-hybrid assay using the 11-mer TALE-VP16 library. A bait sequence was cloned in front of an antibiotic resistance gene (Aba resistance gene) in yeast. The 11-mer TALE-VP16 library was then transformed into this stable clone and a surviving assay was performed on -Leu plates containing 100 nM Aba. (b) The RVD sequences of isolated TALE-VP16 fusions and their targets within the human SCN9A bait sequence (scale not proportional). TALE-VP16 fusions were shown to bind to both the plus and the minus strands of the SCN9A bait sequence. (c) The isolated TALE-VP16 fusions induced overexpression of endogenous SCN9A in HEK293 cells and A431 cells. The mRNA levels of SCN9A were determined by quantitative RT-PCR. An empty vector (PEF-1) was used as the control. Columns 1-6: All TALE-VP16 fusions were able to effectively induce the overexpression of SCN9A in HEK293 cells (n = 5). Columns 11-16: All TALE-VP16 fusions effectively induced the overexpression of SCN9A in A431 cells (n = 3). Columns 7–10: All 4 TALEs designed according to TALE-NT 2.0 failed to induce the overexpression of SCN9A in HEK293 cells (n = 3). Inlet: Western blot shows that all TALE-VP16 fusions induced the overexpression of SCN9A protein in A431 cells (representative data of two independent experiments). (d) The RVD sequences of isolated TALE-VP16 fusions and their targets within the human miR-34b/c bait sequence (scale not proportional). (e) Confirmation of the binding between isolated TALE-VP16 fusion M1 and its predicted gene target within the human miR-34b/c bait sequence. The isolated clone M1 was predicted to target 5′-TTTCTAGGTAT-3′ within the miR-34b/c bait sequence. The full-length bait sequence (pAbAi-miR-34b/c) or bait with the predicted target site deleted (pAbAi-miR-34b/c (ΔTTTCTAGGTAT)) was stably integrated into yeast cells. TALE-VP16 fusion clone M1 was then transformed into either cell line. Only cells which contained the intact bait sequence survived the 100 nM Aba selection. (f) The isolated TALE-VP16 fusion M1 effectively induced overexpression of miR-34b in a dose-dependent manner in both HEK293 and HeLa cells (n = 3 for both cell lines).

    Journal: Scientific Reports

    Article Title: Assembly and Validation of Versatile Transcription Activator-Like Effector Libraries

    doi: 10.1038/srep04857

    Figure Lengend Snippet: Isolation of TALE-VP16 fusions targeting the human SCN9A gene using the 11-mer TALE-VP16 library and the yeast one-hybrid assay. (a) Schematic illustration of the yeast one-hybrid assay using the 11-mer TALE-VP16 library. A bait sequence was cloned in front of an antibiotic resistance gene (Aba resistance gene) in yeast. The 11-mer TALE-VP16 library was then transformed into this stable clone and a surviving assay was performed on -Leu plates containing 100 nM Aba. (b) The RVD sequences of isolated TALE-VP16 fusions and their targets within the human SCN9A bait sequence (scale not proportional). TALE-VP16 fusions were shown to bind to both the plus and the minus strands of the SCN9A bait sequence. (c) The isolated TALE-VP16 fusions induced overexpression of endogenous SCN9A in HEK293 cells and A431 cells. The mRNA levels of SCN9A were determined by quantitative RT-PCR. An empty vector (PEF-1) was used as the control. Columns 1-6: All TALE-VP16 fusions were able to effectively induce the overexpression of SCN9A in HEK293 cells (n = 5). Columns 11-16: All TALE-VP16 fusions effectively induced the overexpression of SCN9A in A431 cells (n = 3). Columns 7–10: All 4 TALEs designed according to TALE-NT 2.0 failed to induce the overexpression of SCN9A in HEK293 cells (n = 3). Inlet: Western blot shows that all TALE-VP16 fusions induced the overexpression of SCN9A protein in A431 cells (representative data of two independent experiments). (d) The RVD sequences of isolated TALE-VP16 fusions and their targets within the human miR-34b/c bait sequence (scale not proportional). (e) Confirmation of the binding between isolated TALE-VP16 fusion M1 and its predicted gene target within the human miR-34b/c bait sequence. The isolated clone M1 was predicted to target 5′-TTTCTAGGTAT-3′ within the miR-34b/c bait sequence. The full-length bait sequence (pAbAi-miR-34b/c) or bait with the predicted target site deleted (pAbAi-miR-34b/c (ΔTTTCTAGGTAT)) was stably integrated into yeast cells. TALE-VP16 fusion clone M1 was then transformed into either cell line. Only cells which contained the intact bait sequence survived the 100 nM Aba selection. (f) The isolated TALE-VP16 fusion M1 effectively induced overexpression of miR-34b in a dose-dependent manner in both HEK293 and HeLa cells (n = 3 for both cell lines).

    Article Snippet: The membranes were then incubated with either anti-SCN9A monoclonal antibody (1:500 dilution in TBS-T, Millipore, #MABN41) or anti-actin monoclonal antibody (1:5,000 dilution in TBS-T, Millipore, #MAB1501) at 4°C overnight.

    Techniques: Isolation, Y1H Assay, Sequencing, Clone Assay, Transformation Assay, Stable Transfection, Over Expression, Quantitative RT-PCR, Plasmid Preparation, Western Blot, Binding Assay, Selection

    Collagen binding by chondroadherin. (A) Binding of biotinylated chondroadherin to immobilised Collagen Toolkit II peptides. Binding was detected with streptavidin-HRP and TMB substrate (absorbance at 450 nm). Error bars show standard deviation from three technical replicates. (B) Dose-response curve of chondroadherin binding to peptide II-26 peptide interaction. (C) Inhibition of the chondroadherin-collagen II interaction by peptide II-26 peptide (solid line) or peptide III-8 (negative control, dashed line). (D) Binding of peptide AB31 to chondroadherin (CHAD) in solution analysed by size exclusion chromatography. AB31 and chondroadherin were mixed in a 2:1 molar ration and incubated for 15 min before injection onto the Superdex 75 column. The sequences of peptides II-26 and AB31 are indicated on the right and the sequence common to both is shaded.

    Journal: Matrix Biology

    Article Title: Structural and functional analysis of two small leucine-rich repeat proteoglycans, fibromodulin and chondroadherin

    doi: 10.1016/j.matbio.2017.02.002

    Figure Lengend Snippet: Collagen binding by chondroadherin. (A) Binding of biotinylated chondroadherin to immobilised Collagen Toolkit II peptides. Binding was detected with streptavidin-HRP and TMB substrate (absorbance at 450 nm). Error bars show standard deviation from three technical replicates. (B) Dose-response curve of chondroadherin binding to peptide II-26 peptide interaction. (C) Inhibition of the chondroadherin-collagen II interaction by peptide II-26 peptide (solid line) or peptide III-8 (negative control, dashed line). (D) Binding of peptide AB31 to chondroadherin (CHAD) in solution analysed by size exclusion chromatography. AB31 and chondroadherin were mixed in a 2:1 molar ration and incubated for 15 min before injection onto the Superdex 75 column. The sequences of peptides II-26 and AB31 are indicated on the right and the sequence common to both is shaded.

    Article Snippet: After rinsing with TBS, plates were incubated with biotinylated chondroadherin at 10 μg/ml in TBST (TBS with 0.1% Tween-20) with 0.1% BSA for 1 h. Plates were washed three times with TBST, and incubated with streptavidin-HRP (Millipore) diluted 1:20,000 in TBST with 0.1% BSA for 1 h. Plates were washed five times with TBST, and the binding was detected with TMB substrate (ThermoFisher) and stopped with 2 M sulphuric acid.

    Techniques: Binding Assay, Standard Deviation, Inhibition, Negative Control, Size-exclusion Chromatography, Incubation, Injection, Sequencing

    Polytene chromosome spreads of  D. melanogaster  wild type were treated with RNase A/RNase H mixture followed by proteinase K digestion in a time course experiment and subsequent immunological detection of triple-stranded DNA. DAPI staining (blue signal) and antibody labelling (red signal) were superimposed. Scale bar represents 25 µm.

    Journal: Cells

    Article Title: Triple-Helical DNA in Drosophila Heterochromatin

    doi: 10.3390/cells7120227

    Figure Lengend Snippet: Polytene chromosome spreads of D. melanogaster wild type were treated with RNase A/RNase H mixture followed by proteinase K digestion in a time course experiment and subsequent immunological detection of triple-stranded DNA. DAPI staining (blue signal) and antibody labelling (red signal) were superimposed. Scale bar represents 25 µm.

    Article Snippet: For RNase treatment, chromosome spreads were rehydrated in 1× TBS followed by incubation at room temperature with RNase A (Calbiochem, San Diego, CA, USA) diluted (0.2 mg/mL) in 2× SSC for 2 h. Additional enzymatic treatments were carried out at room temperature with a mixture of RNase A (Calbiochem, San Diego, CA, USA, 0.2 mg/mL) and RNase H (GE Healthcare, Chicago, IL, USA, 1 unit per slide) diluted in 1× PBS.

    Techniques: Staining