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Millipore tris buffered saline
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% <t>NHS</t> 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 <t>Tris</t> 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
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Images

1) 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

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 "Generation and Characterization of a DNA-GCN4 Oligonucleotide-Peptide Conjugate: The Impact DNA/Protein Interactions on the Sensitization of DNA"

Article Title: Generation and Characterization of a DNA-GCN4 Oligonucleotide-Peptide Conjugate: The Impact DNA/Protein Interactions on the Sensitization of DNA

Journal: Molecules

doi: 10.3390/molecules25163630

Digestion pattern of dsDNA (blue) and dsDNA*-PEP (orange) by DNase I [Charge axis correspond to the following sequence starting from 5′ end—ssDNA* A (5′-GCA CGT CAT CCG TATAG-3′)]. The resulting chromatogram (based on data similar to the PAGE method) is taken from LC-MS analysis. Each peak on TIC (total ion current) was identified and their areas were plotted against the charge of the recognized fragment. The AP-1 sequence corresponds to the 5 to 8 charge fragment. 200 pmol of dsDNA*-PEP or dsDNA was incubated with 0.04 U of DNase I in 10 mM CaCl 2 , 100 mM MgCl 2 , 100 mM Tris-HCl (pH 8.9) buffer solution. The digestion was performed at 4 °C for 10 min.
Figure Legend Snippet: Digestion pattern of dsDNA (blue) and dsDNA*-PEP (orange) by DNase I [Charge axis correspond to the following sequence starting from 5′ end—ssDNA* A (5′-GCA CGT CAT CCG TATAG-3′)]. The resulting chromatogram (based on data similar to the PAGE method) is taken from LC-MS analysis. Each peak on TIC (total ion current) was identified and their areas were plotted against the charge of the recognized fragment. The AP-1 sequence corresponds to the 5 to 8 charge fragment. 200 pmol of dsDNA*-PEP or dsDNA was incubated with 0.04 U of DNase I in 10 mM CaCl 2 , 100 mM MgCl 2 , 100 mM Tris-HCl (pH 8.9) buffer solution. The digestion was performed at 4 °C for 10 min.

Techniques Used: Sequencing, Polyacrylamide Gel Electrophoresis, Liquid Chromatography with Mass Spectroscopy, Incubation

4) 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

5) 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

6) 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

7) Product Images from "α-Synuclein Oligomer Detection with Aptamer Switch on Reduced Graphene Oxide Electrode"

Article Title: α-Synuclein Oligomer Detection with Aptamer Switch on Reduced Graphene Oxide Electrode

Journal: Nanomaterials

doi: 10.3390/nano10050832

( A ) Scanning electron microscopy (SEM) image of ERGO-GCE. ( B ) Contact angle data of water droplets on GCE, ERGO-GCE, and Apt/ERGO-GCE. ( C ) Cyclic voltammetry (CV) curves recorded at a scan rate of 10 mV·s −1 and ( D ) Nyquist plots of GCE, ERGO-GCE, and Apt/ERGO-GCE in 0.1 M tris-buffered saline (TBS) buffer containing 5.0 mM [Fe(CN) 6 ] 3− .
Figure Legend Snippet: ( A ) Scanning electron microscopy (SEM) image of ERGO-GCE. ( B ) Contact angle data of water droplets on GCE, ERGO-GCE, and Apt/ERGO-GCE. ( C ) Cyclic voltammetry (CV) curves recorded at a scan rate of 10 mV·s −1 and ( D ) Nyquist plots of GCE, ERGO-GCE, and Apt/ERGO-GCE in 0.1 M tris-buffered saline (TBS) buffer containing 5.0 mM [Fe(CN) 6 ] 3− .

Techniques Used: Electron Microscopy

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Incubation:

Article Title: Mesoporous silica nanoparticles trigger mitophagy in endothelial cells and perturb neuronal network activity in a size- and time-dependent manner
Article Snippet: Protein bands were transferred to a nitrocellulose membrane by WB and analyzed by immu-noblotting. .. To detect β-actin and caspase 3, the membrane was blocked for 30 minutes at 37°C in Tris-buffered saline containing 0.1% Tween and 5% nonfat milk and incubated overnight at 4°C with anti-β-actin (1:1,500; Sigma-Aldrich) or anti-caspase 3 (1:1,000; Cell Signaling Technology, Beverly, MA, USA). .. For LC3 II, the membrane was blocked for 30 minutes at 37°C in Tris-buffered saline containing 0.1% Tween and 5% bovine serum albumin and incubated overnight at 4°C with anti-LC3 antibody (1:1,000; Cell Signaling Technology).

Article Title: Effect of the Calpain System on Volatile Flavor Compounds in the BeefLongissimus lumborum Muscle
Article Snippet: .. Subsequently, the membrane was blocked using 5% skim milk at room temperature for 1 h. The membrane was incubated with anti-troponin-T monoclonal primary antibodies (1:2,500 in TBS-T (Tris buffered saline containing 0.05% Tween-20), (JLT-12, Sigma-aldrich, USA) and µ-calpain (1:1,000 in TBS-T) (Sigma-aldrich, USA) for 1 h. The bound primary antibodies were labeled with mouse IgG alkaline phosphatase-conjugated secondary antibody (diluted 1:2,500 in TTBS) (Sigma-aldrich, USA) for 1 h. The bound antibodies were visualized using an enhanced chemiluminescence (ECL) kit (Thermo Scientific, USA) and then images were acquired with a Versa Doc 3000 imaging system (Bio-Rad, USA). ..

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
Article Snippet: The cell lysate or conditioned medium was incubated for 4 hours at 4°C with 40 μl of anti–FLAG M2 affinity gel (Sigma). .. The gel was washed in lysis buffer and Tris buffered saline (TBS; 50 mM Tris HCl, pH 7.4, 150 mM NaCl), and bound proteins were eluted by incubation with 150 ng/μl of 3× FLAG peptide (Sigma) in TBS for 30 minutes at 4°C. .. Eluted proteins were recovered by centrifugation, filtered (0.22-μm Ultrafree-MC filter units [Millipore]), and stored at −80°C.

Article Title: Shielding of a Lipooligosaccharide IgM Epitope Allows Evasion of Neutrophil-Mediated Killing of an Invasive Strain of Nontypeable Haemophilus influenzae
Article Snippet: LOS samples were separated on a Tris-Tricine SDS-PAGE gel with a Protean II xi cell electrophoresis system (Bio-Rad) and visualized by silver staining or transferred to a polyvinylidene difluoride (PVDF) membrane for Western blotting. .. Membranes were blocked with 5% bovine serum albumin (BSA) in PBS for 1 h, incubated for 2 h with 5% NHS in PBS, washed 5 times for 5 min (each) with Tris-buffered saline plus 0.05% Tween 20, and incubated with 1:3,000-diluted alkaline phosphatase (AP)-labeled goat-anti-human IgM μ-chain (Sigma-Aldrich). .. Binding was detected with NBT (4-nitroblue tetrazolium ch loride)-BCIP (5-bromo-4-chloro-3-indolyl phosphate) (Roche), following the manufacturer’s instructions.

Labeling:

Article Title: Effect of the Calpain System on Volatile Flavor Compounds in the BeefLongissimus lumborum Muscle
Article Snippet: .. Subsequently, the membrane was blocked using 5% skim milk at room temperature for 1 h. The membrane was incubated with anti-troponin-T monoclonal primary antibodies (1:2,500 in TBS-T (Tris buffered saline containing 0.05% Tween-20), (JLT-12, Sigma-aldrich, USA) and µ-calpain (1:1,000 in TBS-T) (Sigma-aldrich, USA) for 1 h. The bound primary antibodies were labeled with mouse IgG alkaline phosphatase-conjugated secondary antibody (diluted 1:2,500 in TTBS) (Sigma-aldrich, USA) for 1 h. The bound antibodies were visualized using an enhanced chemiluminescence (ECL) kit (Thermo Scientific, USA) and then images were acquired with a Versa Doc 3000 imaging system (Bio-Rad, USA). ..

Imaging:

Article Title: Effect of the Calpain System on Volatile Flavor Compounds in the BeefLongissimus lumborum Muscle
Article Snippet: .. Subsequently, the membrane was blocked using 5% skim milk at room temperature for 1 h. The membrane was incubated with anti-troponin-T monoclonal primary antibodies (1:2,500 in TBS-T (Tris buffered saline containing 0.05% Tween-20), (JLT-12, Sigma-aldrich, USA) and µ-calpain (1:1,000 in TBS-T) (Sigma-aldrich, USA) for 1 h. The bound primary antibodies were labeled with mouse IgG alkaline phosphatase-conjugated secondary antibody (diluted 1:2,500 in TTBS) (Sigma-aldrich, USA) for 1 h. The bound antibodies were visualized using an enhanced chemiluminescence (ECL) kit (Thermo Scientific, USA) and then images were acquired with a Versa Doc 3000 imaging system (Bio-Rad, USA). ..

other:

Article Title: Running in pregnancy transiently increases postnatal hippocampal neurogenesis in the offspring
Article Snippet: Total granule-cell numbers were determined in sections treated with DNA-stain bisbenzemide (Hoechst 33258; Sigma; 50 ng/ml Tris-buffered saline for 15 min).

Lysis:

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
Article Snippet: The cell lysate or conditioned medium was incubated for 4 hours at 4°C with 40 μl of anti–FLAG M2 affinity gel (Sigma). .. The gel was washed in lysis buffer and Tris buffered saline (TBS; 50 mM Tris HCl, pH 7.4, 150 mM NaCl), and bound proteins were eluted by incubation with 150 ng/μl of 3× FLAG peptide (Sigma) in TBS for 30 minutes at 4°C. .. Eluted proteins were recovered by centrifugation, filtered (0.22-μm Ultrafree-MC filter units [Millipore]), and stored at −80°C.

Western Blot:

Article Title: The RhoGAP protein ARHGAP18/SENEX localizes to microtubules and regulates their stability in endothelial cells
Article Snippet: .. Western blot membranes were probed for α-tubulin (DM1A clone, Sigma-Aldrich; primary antibody, 1:10,000 in 0.1% skim milk/Tris-buffered saline/Tween 20 [TBST] overnight; secondary antibody, 1:10,000 goat anti-mouse HRP-conjugated antibody in 0.1% skim milk/TBST for 1 h at room temperature) and ARHGAP18 (primary antibody, 1:10,000 or 1:1000 in 5% BSA/TBST; secondary antibody, 1:10,000 or 1:1,000 anti-mouse HRP-conjugated antibody in 5% BSA/TBST for 1 h at room temperature) and visualized using Pierce ECL Prime and x-ray development. .. Western blot densitometry was performed using ImageJ.

Transfection:

Article Title: Association with the Cellular Export Receptor CRM 1 Mediates Function and Intracellular Localization of Epstein-Barr Virus SM Protein, a Regulator of Gene Expression
Article Snippet: Cos 7 cells were transfected with LipofectaminePlus, per the manufacturer’s protocol (Gibco Life Sciences). .. Cells were lysed 48 h after transfection in immunoprecipitation buffer (Tris-buffered saline [pH 7.4], 1% Triton X-100, 1 mM dithiothreitol, 100 μM GTP-γS, and a mixture of protease inhibitors [Sigma protease inhibitor cocktail no. P2714]). .. One hundred fifty microliters of each lysate was cleared with preimmune serum, incubated with 1 μl of undiluted polyclonal antibody and 20 μl of protein A-conjugated agarose beads (Sigma) for 90 min at 4°C, and washed four times in immunoprecipitation buffer.

Immunoprecipitation:

Article Title: Association with the Cellular Export Receptor CRM 1 Mediates Function and Intracellular Localization of Epstein-Barr Virus SM Protein, a Regulator of Gene Expression
Article Snippet: Cos 7 cells were transfected with LipofectaminePlus, per the manufacturer’s protocol (Gibco Life Sciences). .. Cells were lysed 48 h after transfection in immunoprecipitation buffer (Tris-buffered saline [pH 7.4], 1% Triton X-100, 1 mM dithiothreitol, 100 μM GTP-γS, and a mixture of protease inhibitors [Sigma protease inhibitor cocktail no. P2714]). .. One hundred fifty microliters of each lysate was cleared with preimmune serum, incubated with 1 μl of undiluted polyclonal antibody and 20 μl of protein A-conjugated agarose beads (Sigma) for 90 min at 4°C, and washed four times in immunoprecipitation buffer.

Protease Inhibitor:

Article Title: Association with the Cellular Export Receptor CRM 1 Mediates Function and Intracellular Localization of Epstein-Barr Virus SM Protein, a Regulator of Gene Expression
Article Snippet: Cos 7 cells were transfected with LipofectaminePlus, per the manufacturer’s protocol (Gibco Life Sciences). .. Cells were lysed 48 h after transfection in immunoprecipitation buffer (Tris-buffered saline [pH 7.4], 1% Triton X-100, 1 mM dithiothreitol, 100 μM GTP-γS, and a mixture of protease inhibitors [Sigma protease inhibitor cocktail no. P2714]). .. One hundred fifty microliters of each lysate was cleared with preimmune serum, incubated with 1 μl of undiluted polyclonal antibody and 20 μl of protein A-conjugated agarose beads (Sigma) for 90 min at 4°C, and washed four times in immunoprecipitation buffer.

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  • 93
    Millipore ifnγ
    Optimization of cell concentration. Milliplex kits from Millipore (see Material and Methods) were used to perform cell concentration optimization experiments using 0.5 or 1.0 million Non-Stimulated (NS) or PPD-Stimulated PBMC from two healthy donors cultured in triplicate for 2 or 5 days. Results obtained with samples from donor 1 (IL-2 and IL-17) and donor 2 <t>(IFNγ</t> and IL-5) are shown at the optimal time point defined for each cytokine. Mean cytokine concentrations are represented by black lines
    Ifnγ, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Millipore anti flag m2 affinity gel
    HSP90 interacts with RUBV p150. (A) A schematic diagram of SINV and CHIKV nsPs. nsP1, nsP2, nsP3, and nsP4 were processed from the precursor polyprotein, nsP1-4, by the viral protease (Pro), located in the nsP2 region. (B) A schematic diagram of RUBV NSPs. p150 and p90 were processed from the precursor polyprotein, p200, by viral protease, located in the p150 region. (C and D) Co-IPA of FLAG HSP90α with SINV 3Myc nsPs (C) and co-IPA of SINV 3Myc nsP4 with FLAG HSP90α (D). (C) 293T cells were cotransfected with plasmids expressing FLAG-tagged HSP90α ( FLAG HSP90α) and Myc-tagged SINV nsPs (SINV 3Myc nsPs). SINV 3Myc nsPs were immunoprecipitated with anti-Myc MAb. Proteins in the total cell lysate (input) and in the immunoprecipitated (IP) complexes were detected by immunoblotting using <t>anti-FLAG,</t> anti-Myc, and anti-GAPDH MAbs. (D) For immunoprecipitation with FLAG HSP90α, 293T cells were cotransfected with plasmids expressing FLAG HSP90α and SINV 3Myc nsP4. FLAG HSP90α was immunoprecipitated with an anti-FLAG MAb. Proteins in the total cell lysate (input) and in the immunoprecipitated complexes were detected by immunoblotting using anti-FLAG, anti-Myc, and anti-GAPDH MAbs. (E and F) Co-IPA of FLAG HSP90α with CHIKV 3Myc nsPs (E) and co-IPA of CHIKV 3Myc nsP4 with FLAG HSP90α (F). These co-IPA experiments were performed as described in the legend to panels C and D using CHIKV 3Myc nsPs instead of SINV 3Myc nsPs. (G and H) Co-IPA of RUBV NSPs with FLAG HSP90α (G) and co-IPA of FLAG HSP90α with RUBV p200 or p150 (H). (G) 293T cells were cotransfected with plasmids expressing AG1-tagged NSP ( AG1 p200 C1152G , AG1 p150, or AG1 p90) and FLAG-tagged FLAG HSP90α. FLAG HSP90α was immunoprecipitated with an anti-FLAG M2 affinity gel, and the proteins in the total cell lysate (input) and in the immunoprecipitated complexes were detected by immunoblotting using an anti-FLAG pAb, an anti-AG1 pAb, and an anti-GAPDH MAb. (H) For immunoprecipitation with AG1 p200 C1152G or AG1 p150, 293T cells were transfected with plasmids expressing AG1-tagged NSP ( AG1 p200 C1152G or AG1 p150) and FLAG HSP90α. AG1 p200 C1152G or AG1 p150 was immunoprecipitated with an anti-AG1 pAb. Proteins in the total cell lysate (input) and in the immunoprecipitated complexes were detected by immunoblotting using an anti-AG1 pAb, an anti-FLAG MAb, and an anti-GAPDH MAb. (C to H) Data are representative of those from three independent experiments.
    Anti Flag M2 Affinity Gel, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore goat anti mouse igg polyclonal antibodies conjugated
    Concentration of <t>IgG</t> and S-IgA. The concentrations of IgG ( a ) and S-IgA ( b ) in seminal plasmas were determined by radial immunodiffusion [ 22 ] and immunoenzymatic sandwich ELISA test, using goat anti-human IgG <t>polyclonal</t> antibodies and mouse anti-human SC of IgA monoclonal antibodies, respectively
    Goat Anti Mouse Igg Polyclonal Antibodies Conjugated, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Millipore 4 6 diamidino 2 phenylindole dapi
    Localization of Slc26a4 and temporal changes in Slc26a4 and SCN − levels in O 3 -exposed mice, and the inhibitory effects of NH 4 Cl. ( A ) Representative SCN − in mice BALF of sham, O 3 -exposed, and NH 4 Cl-treated O 3 -exposed mice on Day 21. ( B ) Representative western blot of Slc26a4 in lung lysates pooled from sham (n = 6), O 3 -exposed (n = 6), and NH 4 Cl-treated O 3 -exposed (n = 6) mice on Day 21. Data represent the mean ± SEM of six experiments and P values were determined using t -tests. ( C ) Representative image of H E and immunofluorescence staining for Slc26a4 (green, FITC) and Muc5ac (red, PE) in lung tissues of sham, O 3 -exposed, and NH 4 Cl-treated O 3 -exposed mice on Day 21. Nuclei were stained with DAPI (blue) (1,000×). Slc26a4 = solute carrier family 26 member 4, SCN − = thiocyanate, O 3 = ozone, NH 4 Cl = ammonium chloride, SEM = standard error of the mean, H E = hematoxylin eosin, Muc5ac = mucin 5AC, DAPI = <t>4′,6-diamidino-2-phenylindole,</t> A = alveoli, B = bronchiole. * P
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    Image Search Results


    Optimization of cell concentration. Milliplex kits from Millipore (see Material and Methods) were used to perform cell concentration optimization experiments using 0.5 or 1.0 million Non-Stimulated (NS) or PPD-Stimulated PBMC from two healthy donors cultured in triplicate for 2 or 5 days. Results obtained with samples from donor 1 (IL-2 and IL-17) and donor 2 (IFNγ and IL-5) are shown at the optimal time point defined for each cytokine. Mean cytokine concentrations are represented by black lines

    Journal: BMC Immunology

    Article Title: Optimization and evaluation of Luminex performance with supernatants of antigen-stimulated peripheral blood mononuclear cells

    doi: 10.1186/s12865-016-0182-8

    Figure Lengend Snippet: Optimization of cell concentration. Milliplex kits from Millipore (see Material and Methods) were used to perform cell concentration optimization experiments using 0.5 or 1.0 million Non-Stimulated (NS) or PPD-Stimulated PBMC from two healthy donors cultured in triplicate for 2 or 5 days. Results obtained with samples from donor 1 (IL-2 and IL-17) and donor 2 (IFNγ and IL-5) are shown at the optimal time point defined for each cytokine. Mean cytokine concentrations are represented by black lines

    Article Snippet: SD calculated using culture triplicates for NS, PPD- and SEB-stimulated PBMC from TB-infected patient plotted against IFNγ, IL-5 and IL-17A corresponding mean concentration values for Ozyme, Millipore and Bio-Rad kits. (PDF 14 kb)

    Techniques: Concentration Assay, Cell Culture

    Kinetics of cytokine production. Milliplex kits from Millipore (see Material and Methods for details) were used to perform kinetic experiments with PBMC from two healthy donors stimulated for 1, 2 or 5 days with PPD (1 μg/ml) and SEB (10 ng/ml) as positive control. Non-stimulated (NS) PBMC were used as negative control. Results obtained for samples from donor 1 (IL-2 and IL-17) and donor 2 (IFNγ and IL-5) are shown. NS and PPD values correspond to the left y axis and SEB values to the right y axis

    Journal: BMC Immunology

    Article Title: Optimization and evaluation of Luminex performance with supernatants of antigen-stimulated peripheral blood mononuclear cells

    doi: 10.1186/s12865-016-0182-8

    Figure Lengend Snippet: Kinetics of cytokine production. Milliplex kits from Millipore (see Material and Methods for details) were used to perform kinetic experiments with PBMC from two healthy donors stimulated for 1, 2 or 5 days with PPD (1 μg/ml) and SEB (10 ng/ml) as positive control. Non-stimulated (NS) PBMC were used as negative control. Results obtained for samples from donor 1 (IL-2 and IL-17) and donor 2 (IFNγ and IL-5) are shown. NS and PPD values correspond to the left y axis and SEB values to the right y axis

    Article Snippet: SD calculated using culture triplicates for NS, PPD- and SEB-stimulated PBMC from TB-infected patient plotted against IFNγ, IL-5 and IL-17A corresponding mean concentration values for Ozyme, Millipore and Bio-Rad kits. (PDF 14 kb)

    Techniques: Positive Control, Negative Control

    HSP90 interacts with RUBV p150. (A) A schematic diagram of SINV and CHIKV nsPs. nsP1, nsP2, nsP3, and nsP4 were processed from the precursor polyprotein, nsP1-4, by the viral protease (Pro), located in the nsP2 region. (B) A schematic diagram of RUBV NSPs. p150 and p90 were processed from the precursor polyprotein, p200, by viral protease, located in the p150 region. (C and D) Co-IPA of FLAG HSP90α with SINV 3Myc nsPs (C) and co-IPA of SINV 3Myc nsP4 with FLAG HSP90α (D). (C) 293T cells were cotransfected with plasmids expressing FLAG-tagged HSP90α ( FLAG HSP90α) and Myc-tagged SINV nsPs (SINV 3Myc nsPs). SINV 3Myc nsPs were immunoprecipitated with anti-Myc MAb. Proteins in the total cell lysate (input) and in the immunoprecipitated (IP) complexes were detected by immunoblotting using anti-FLAG, anti-Myc, and anti-GAPDH MAbs. (D) For immunoprecipitation with FLAG HSP90α, 293T cells were cotransfected with plasmids expressing FLAG HSP90α and SINV 3Myc nsP4. FLAG HSP90α was immunoprecipitated with an anti-FLAG MAb. Proteins in the total cell lysate (input) and in the immunoprecipitated complexes were detected by immunoblotting using anti-FLAG, anti-Myc, and anti-GAPDH MAbs. (E and F) Co-IPA of FLAG HSP90α with CHIKV 3Myc nsPs (E) and co-IPA of CHIKV 3Myc nsP4 with FLAG HSP90α (F). These co-IPA experiments were performed as described in the legend to panels C and D using CHIKV 3Myc nsPs instead of SINV 3Myc nsPs. (G and H) Co-IPA of RUBV NSPs with FLAG HSP90α (G) and co-IPA of FLAG HSP90α with RUBV p200 or p150 (H). (G) 293T cells were cotransfected with plasmids expressing AG1-tagged NSP ( AG1 p200 C1152G , AG1 p150, or AG1 p90) and FLAG-tagged FLAG HSP90α. FLAG HSP90α was immunoprecipitated with an anti-FLAG M2 affinity gel, and the proteins in the total cell lysate (input) and in the immunoprecipitated complexes were detected by immunoblotting using an anti-FLAG pAb, an anti-AG1 pAb, and an anti-GAPDH MAb. (H) For immunoprecipitation with AG1 p200 C1152G or AG1 p150, 293T cells were transfected with plasmids expressing AG1-tagged NSP ( AG1 p200 C1152G or AG1 p150) and FLAG HSP90α. AG1 p200 C1152G or AG1 p150 was immunoprecipitated with an anti-AG1 pAb. Proteins in the total cell lysate (input) and in the immunoprecipitated complexes were detected by immunoblotting using an anti-AG1 pAb, an anti-FLAG MAb, and an anti-GAPDH MAb. (C to H) Data are representative of those from three independent experiments.

    Journal: Journal of Virology

    Article Title: Heat Shock Protein 90 Ensures the Integrity of Rubella Virus p150 Protein and Supports Viral Replication

    doi: 10.1128/JVI.01142-19

    Figure Lengend Snippet: HSP90 interacts with RUBV p150. (A) A schematic diagram of SINV and CHIKV nsPs. nsP1, nsP2, nsP3, and nsP4 were processed from the precursor polyprotein, nsP1-4, by the viral protease (Pro), located in the nsP2 region. (B) A schematic diagram of RUBV NSPs. p150 and p90 were processed from the precursor polyprotein, p200, by viral protease, located in the p150 region. (C and D) Co-IPA of FLAG HSP90α with SINV 3Myc nsPs (C) and co-IPA of SINV 3Myc nsP4 with FLAG HSP90α (D). (C) 293T cells were cotransfected with plasmids expressing FLAG-tagged HSP90α ( FLAG HSP90α) and Myc-tagged SINV nsPs (SINV 3Myc nsPs). SINV 3Myc nsPs were immunoprecipitated with anti-Myc MAb. Proteins in the total cell lysate (input) and in the immunoprecipitated (IP) complexes were detected by immunoblotting using anti-FLAG, anti-Myc, and anti-GAPDH MAbs. (D) For immunoprecipitation with FLAG HSP90α, 293T cells were cotransfected with plasmids expressing FLAG HSP90α and SINV 3Myc nsP4. FLAG HSP90α was immunoprecipitated with an anti-FLAG MAb. Proteins in the total cell lysate (input) and in the immunoprecipitated complexes were detected by immunoblotting using anti-FLAG, anti-Myc, and anti-GAPDH MAbs. (E and F) Co-IPA of FLAG HSP90α with CHIKV 3Myc nsPs (E) and co-IPA of CHIKV 3Myc nsP4 with FLAG HSP90α (F). These co-IPA experiments were performed as described in the legend to panels C and D using CHIKV 3Myc nsPs instead of SINV 3Myc nsPs. (G and H) Co-IPA of RUBV NSPs with FLAG HSP90α (G) and co-IPA of FLAG HSP90α with RUBV p200 or p150 (H). (G) 293T cells were cotransfected with plasmids expressing AG1-tagged NSP ( AG1 p200 C1152G , AG1 p150, or AG1 p90) and FLAG-tagged FLAG HSP90α. FLAG HSP90α was immunoprecipitated with an anti-FLAG M2 affinity gel, and the proteins in the total cell lysate (input) and in the immunoprecipitated complexes were detected by immunoblotting using an anti-FLAG pAb, an anti-AG1 pAb, and an anti-GAPDH MAb. (H) For immunoprecipitation with AG1 p200 C1152G or AG1 p150, 293T cells were transfected with plasmids expressing AG1-tagged NSP ( AG1 p200 C1152G or AG1 p150) and FLAG HSP90α. AG1 p200 C1152G or AG1 p150 was immunoprecipitated with an anti-AG1 pAb. Proteins in the total cell lysate (input) and in the immunoprecipitated complexes were detected by immunoblotting using an anti-AG1 pAb, an anti-FLAG MAb, and an anti-GAPDH MAb. (C to H) Data are representative of those from three independent experiments.

    Article Snippet: After being washed with TBS-T five times, the proteins bound to the anti-FLAG M2 affinity gel were dissolved by boiling the samples with SDS sample buffer for 5 min.

    Techniques: Indirect Immunoperoxidase Assay, Expressing, Immunoprecipitation, Transfection

    Concentration of IgG and S-IgA. The concentrations of IgG ( a ) and S-IgA ( b ) in seminal plasmas were determined by radial immunodiffusion [ 22 ] and immunoenzymatic sandwich ELISA test, using goat anti-human IgG polyclonal antibodies and mouse anti-human SC of IgA monoclonal antibodies, respectively

    Journal: Glycoconjugate Journal

    Article Title: Changes in fucosylation of human seminal IgG and secretory component of IgA in leukocytospermic patients

    doi: 10.1007/s10719-013-9501-y

    Figure Lengend Snippet: Concentration of IgG and S-IgA. The concentrations of IgG ( a ) and S-IgA ( b ) in seminal plasmas were determined by radial immunodiffusion [ 22 ] and immunoenzymatic sandwich ELISA test, using goat anti-human IgG polyclonal antibodies and mouse anti-human SC of IgA monoclonal antibodies, respectively

    Article Snippet: After blocking (3 % powdered skim milk in 50 mM TBS, pH 7.5), the blots were incubated with mouse monoclonal antibodies anti-human SC of IgA diluted at 1:1000 in 3 % powdered skim milk in 50 mM TBS, pH 7.5, and probed with goat anti-mouse IgG polyclonal antibodies conjugated with horseradish peroxidase (1:2000 dilution in 3 % powdered skim milk dissolved in 50 mM TBS; Sigma Chemical Co., St. Louis, MO, USA).

    Techniques: Concentration Assay, Radial Immuno Diffusion, Sandwich ELISA

    Localization of Slc26a4 and temporal changes in Slc26a4 and SCN − levels in O 3 -exposed mice, and the inhibitory effects of NH 4 Cl. ( A ) Representative SCN − in mice BALF of sham, O 3 -exposed, and NH 4 Cl-treated O 3 -exposed mice on Day 21. ( B ) Representative western blot of Slc26a4 in lung lysates pooled from sham (n = 6), O 3 -exposed (n = 6), and NH 4 Cl-treated O 3 -exposed (n = 6) mice on Day 21. Data represent the mean ± SEM of six experiments and P values were determined using t -tests. ( C ) Representative image of H E and immunofluorescence staining for Slc26a4 (green, FITC) and Muc5ac (red, PE) in lung tissues of sham, O 3 -exposed, and NH 4 Cl-treated O 3 -exposed mice on Day 21. Nuclei were stained with DAPI (blue) (1,000×). Slc26a4 = solute carrier family 26 member 4, SCN − = thiocyanate, O 3 = ozone, NH 4 Cl = ammonium chloride, SEM = standard error of the mean, H E = hematoxylin eosin, Muc5ac = mucin 5AC, DAPI = 4′,6-diamidino-2-phenylindole, A = alveoli, B = bronchiole. * P

    Journal: Journal of Korean Medical Science

    Article Title: Effects of Ammonium Chloride on Ozone-induced Airway Inflammation: the Role of Slc26a4 in the Lungs of Mice

    doi: 10.3346/jkms.2020.35.e272

    Figure Lengend Snippet: Localization of Slc26a4 and temporal changes in Slc26a4 and SCN − levels in O 3 -exposed mice, and the inhibitory effects of NH 4 Cl. ( A ) Representative SCN − in mice BALF of sham, O 3 -exposed, and NH 4 Cl-treated O 3 -exposed mice on Day 21. ( B ) Representative western blot of Slc26a4 in lung lysates pooled from sham (n = 6), O 3 -exposed (n = 6), and NH 4 Cl-treated O 3 -exposed (n = 6) mice on Day 21. Data represent the mean ± SEM of six experiments and P values were determined using t -tests. ( C ) Representative image of H E and immunofluorescence staining for Slc26a4 (green, FITC) and Muc5ac (red, PE) in lung tissues of sham, O 3 -exposed, and NH 4 Cl-treated O 3 -exposed mice on Day 21. Nuclei were stained with DAPI (blue) (1,000×). Slc26a4 = solute carrier family 26 member 4, SCN − = thiocyanate, O 3 = ozone, NH 4 Cl = ammonium chloride, SEM = standard error of the mean, H E = hematoxylin eosin, Muc5ac = mucin 5AC, DAPI = 4′,6-diamidino-2-phenylindole, A = alveoli, B = bronchiole. * P

    Article Snippet: After washing in TBS, the slides were incubated with 4′,6-diamidino-2-phenylindole (DAPI) (1:1,000 dilution; Sigma-Aldrich).

    Techniques: Mouse Assay, Western Blot, Immunofluorescence, Staining