s-6100  (Echelon Biosciences)


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    Echelon Biosciences s-6100
    S 6100, supplied by Echelon Biosciences, 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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    phosphatidylethanolamine  (Echelon Biosciences)


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    Echelon Biosciences phosphatidylethanolamine
    Sphingosine binds to ACE2 and thereby blocks the interaction of ACE2 with the viral spike protein. A , sphingosine (SPH)-coated beads or control ( Ctr ) beads were incubated with recombinant ( rec. ) Fc-ACE2 protein ( left panel ) or with lysates obtained from Vero cells ( right panel ), extensively washed, and eluted in 1× SDS sample buffer. As indicated, suspended sphingosine ( SPH ) was added (2 μ m ) to prevent binding of ACE2 to immobilized sphingosine. The samples were separated by 7.5% SDS-PAGE electrophoresis and blotted with anti-ACE2 antibodies. The Fc-ACE2 protein has a molecular mass of ∼180 kDa, and the endogenous ACE2 protein has a molecular mass of ∼120 kDa. Shown are representative results from five independent experiments. B , recombinant Fc-ACE2 protein was immobilized on protein A/G–agarose, washed, and incubated with 2 μ m sphingosine. Controls consisted of protein A/G–agarose only, incubated with 2 μ m sphingosine. The samples were washed and extracted in CHCl 3 :CH 3 OH:1N HCl (100:100:1, v/v/v), and sphingosine was quantified employing a kinase assay. Given are the means ± S.D. of the sphingosine concentrations from six independent experiments. ***, p < 0.001, ANOVA followed by post hoc Student's t tests. C , a panel of lipid-coated beads was used to test for the specificity of ACE2-binding to sphingosine-coupled beads. The samples were prepared as above. Shown is a representative result from four independent experiments. D , recombinant Fc-ACE2 protein was immobilized on protein A/G–agarose, washed, incubated with 2 μ m sphingosine or left untreated, washed again, and incubated with the recombinant receptor-binding domain of the spike protein. The samples were washed extensively, eluted, separated by 7.5% SDS-PAGE electrophoresis, blotted, and developed with anti-spike antibodies. The recombinant receptor-binding domain of the spike protein is His-tagged and has a molecular mass of ∼27 kDa. Protein A/G–agarose beads without Fc-ACE2 served as control. Shown are representative results from five independent experiments. E , a variety of other lipids were added as indicated to immobilized recombinant human Fc-ACE2. The effects of these lipids on binding of recombinant receptor-binding domain of the spike protein were determined by Western blotting. Shown is a representative result from four independent studies. F , recombinant His-tagged RBD of spike was immobilized on Ni 2+ –agarose, washed, incubated with 2 μ m sphingosine, or left untreated. Recombinant human Fc-ACE2 was added, and the samples were incubated for 60 min. Beads without the addition of RBD spike served as control. The samples were washed extensively, eluted, separated by 7.5% SDS-PAGE electrophoresis, blotted, and developed with anti-ACE2 antibodies. Shown are representative results from five independent experiments. G , a variety of other lipids were added as indicated to immobilized recombinant RBD–spike protein. The effects of these lipids on binding of recombinant human Fc-ACE2 were determined by Western blotting. Shown is a representative result from four independent studies. PC , phosphatidylcholine; PE , <t>phosphatidylethanolamine;</t> SM , sphingomyelin; CER , ceramide; S1P , sphingosine 1-phosphate; Clp , cardiolipin; PS , phosphatidylserine; C16-CER , C16-ceramide; LC , lactosylceramide; OGP , octylglucopyranoside.
    Phosphatidylethanolamine, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Sphingosine prevents binding of SARS–CoV-2 spike to its cellular receptor ACE2"

    Article Title: Sphingosine prevents binding of SARS–CoV-2 spike to its cellular receptor ACE2

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA120.015249

    Sphingosine binds to ACE2 and thereby blocks the interaction of ACE2 with the viral spike protein. A , sphingosine (SPH)-coated beads or control ( Ctr ) beads were incubated with recombinant ( rec. ) Fc-ACE2 protein ( left panel ) or with lysates obtained from Vero cells ( right panel ), extensively washed, and eluted in 1× SDS sample buffer. As indicated, suspended sphingosine ( SPH ) was added (2 μ m ) to prevent binding of ACE2 to immobilized sphingosine. The samples were separated by 7.5% SDS-PAGE electrophoresis and blotted with anti-ACE2 antibodies. The Fc-ACE2 protein has a molecular mass of ∼180 kDa, and the endogenous ACE2 protein has a molecular mass of ∼120 kDa. Shown are representative results from five independent experiments. B , recombinant Fc-ACE2 protein was immobilized on protein A/G–agarose, washed, and incubated with 2 μ m sphingosine. Controls consisted of protein A/G–agarose only, incubated with 2 μ m sphingosine. The samples were washed and extracted in CHCl 3 :CH 3 OH:1N HCl (100:100:1, v/v/v), and sphingosine was quantified employing a kinase assay. Given are the means ± S.D. of the sphingosine concentrations from six independent experiments. ***, p < 0.001, ANOVA followed by post hoc Student's t tests. C , a panel of lipid-coated beads was used to test for the specificity of ACE2-binding to sphingosine-coupled beads. The samples were prepared as above. Shown is a representative result from four independent experiments. D , recombinant Fc-ACE2 protein was immobilized on protein A/G–agarose, washed, incubated with 2 μ m sphingosine or left untreated, washed again, and incubated with the recombinant receptor-binding domain of the spike protein. The samples were washed extensively, eluted, separated by 7.5% SDS-PAGE electrophoresis, blotted, and developed with anti-spike antibodies. The recombinant receptor-binding domain of the spike protein is His-tagged and has a molecular mass of ∼27 kDa. Protein A/G–agarose beads without Fc-ACE2 served as control. Shown are representative results from five independent experiments. E , a variety of other lipids were added as indicated to immobilized recombinant human Fc-ACE2. The effects of these lipids on binding of recombinant receptor-binding domain of the spike protein were determined by Western blotting. Shown is a representative result from four independent studies. F , recombinant His-tagged RBD of spike was immobilized on Ni 2+ –agarose, washed, incubated with 2 μ m sphingosine, or left untreated. Recombinant human Fc-ACE2 was added, and the samples were incubated for 60 min. Beads without the addition of RBD spike served as control. The samples were washed extensively, eluted, separated by 7.5% SDS-PAGE electrophoresis, blotted, and developed with anti-ACE2 antibodies. Shown are representative results from five independent experiments. G , a variety of other lipids were added as indicated to immobilized recombinant RBD–spike protein. The effects of these lipids on binding of recombinant human Fc-ACE2 were determined by Western blotting. Shown is a representative result from four independent studies. PC , phosphatidylcholine; PE , phosphatidylethanolamine; SM , sphingomyelin; CER , ceramide; S1P , sphingosine 1-phosphate; Clp , cardiolipin; PS , phosphatidylserine; C16-CER , C16-ceramide; LC , lactosylceramide; OGP , octylglucopyranoside.
    Figure Legend Snippet: Sphingosine binds to ACE2 and thereby blocks the interaction of ACE2 with the viral spike protein. A , sphingosine (SPH)-coated beads or control ( Ctr ) beads were incubated with recombinant ( rec. ) Fc-ACE2 protein ( left panel ) or with lysates obtained from Vero cells ( right panel ), extensively washed, and eluted in 1× SDS sample buffer. As indicated, suspended sphingosine ( SPH ) was added (2 μ m ) to prevent binding of ACE2 to immobilized sphingosine. The samples were separated by 7.5% SDS-PAGE electrophoresis and blotted with anti-ACE2 antibodies. The Fc-ACE2 protein has a molecular mass of ∼180 kDa, and the endogenous ACE2 protein has a molecular mass of ∼120 kDa. Shown are representative results from five independent experiments. B , recombinant Fc-ACE2 protein was immobilized on protein A/G–agarose, washed, and incubated with 2 μ m sphingosine. Controls consisted of protein A/G–agarose only, incubated with 2 μ m sphingosine. The samples were washed and extracted in CHCl 3 :CH 3 OH:1N HCl (100:100:1, v/v/v), and sphingosine was quantified employing a kinase assay. Given are the means ± S.D. of the sphingosine concentrations from six independent experiments. ***, p < 0.001, ANOVA followed by post hoc Student's t tests. C , a panel of lipid-coated beads was used to test for the specificity of ACE2-binding to sphingosine-coupled beads. The samples were prepared as above. Shown is a representative result from four independent experiments. D , recombinant Fc-ACE2 protein was immobilized on protein A/G–agarose, washed, incubated with 2 μ m sphingosine or left untreated, washed again, and incubated with the recombinant receptor-binding domain of the spike protein. The samples were washed extensively, eluted, separated by 7.5% SDS-PAGE electrophoresis, blotted, and developed with anti-spike antibodies. The recombinant receptor-binding domain of the spike protein is His-tagged and has a molecular mass of ∼27 kDa. Protein A/G–agarose beads without Fc-ACE2 served as control. Shown are representative results from five independent experiments. E , a variety of other lipids were added as indicated to immobilized recombinant human Fc-ACE2. The effects of these lipids on binding of recombinant receptor-binding domain of the spike protein were determined by Western blotting. Shown is a representative result from four independent studies. F , recombinant His-tagged RBD of spike was immobilized on Ni 2+ –agarose, washed, incubated with 2 μ m sphingosine, or left untreated. Recombinant human Fc-ACE2 was added, and the samples were incubated for 60 min. Beads without the addition of RBD spike served as control. The samples were washed extensively, eluted, separated by 7.5% SDS-PAGE electrophoresis, blotted, and developed with anti-ACE2 antibodies. Shown are representative results from five independent experiments. G , a variety of other lipids were added as indicated to immobilized recombinant RBD–spike protein. The effects of these lipids on binding of recombinant human Fc-ACE2 were determined by Western blotting. Shown is a representative result from four independent studies. PC , phosphatidylcholine; PE , phosphatidylethanolamine; SM , sphingomyelin; CER , ceramide; S1P , sphingosine 1-phosphate; Clp , cardiolipin; PS , phosphatidylserine; C16-CER , C16-ceramide; LC , lactosylceramide; OGP , octylglucopyranoside.

    Techniques Used: Incubation, Recombinant, Binding Assay, SDS Page, Electrophoresis, Kinase Assay, Western Blot

    phosphatidylcholine  (Echelon Biosciences)


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    agarose sphingosine  (Echelon Biosciences)


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    Echelon Biosciences agarose sphingosine
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    s-6100  (Echelon Biosciences)


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    Echelon Biosciences s-6100
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    sphingosine  (Echelon Biosciences)


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    Echelon Biosciences sphingosine
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    sphingosine  (Echelon Biosciences)


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    Echelon Biosciences sphingosine
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    Echelon Biosciences phosphatidylethanolamine
    Sphingosine binds to ACE2 and thereby blocks the interaction of ACE2 with the viral spike protein. A , sphingosine (SPH)-coated beads or control ( Ctr ) beads were incubated with recombinant ( rec. ) Fc-ACE2 protein ( left panel ) or with lysates obtained from Vero cells ( right panel ), extensively washed, and eluted in 1× SDS sample buffer. As indicated, suspended sphingosine ( SPH ) was added (2 μ m ) to prevent binding of ACE2 to immobilized sphingosine. The samples were separated by 7.5% SDS-PAGE electrophoresis and blotted with anti-ACE2 antibodies. The Fc-ACE2 protein has a molecular mass of ∼180 kDa, and the endogenous ACE2 protein has a molecular mass of ∼120 kDa. Shown are representative results from five independent experiments. B , recombinant Fc-ACE2 protein was immobilized on protein A/G–agarose, washed, and incubated with 2 μ m sphingosine. Controls consisted of protein A/G–agarose only, incubated with 2 μ m sphingosine. The samples were washed and extracted in CHCl 3 :CH 3 OH:1N HCl (100:100:1, v/v/v), and sphingosine was quantified employing a kinase assay. Given are the means ± S.D. of the sphingosine concentrations from six independent experiments. ***, p < 0.001, ANOVA followed by post hoc Student's t tests. C , a panel of lipid-coated beads was used to test for the specificity of ACE2-binding to sphingosine-coupled beads. The samples were prepared as above. Shown is a representative result from four independent experiments. D , recombinant Fc-ACE2 protein was immobilized on protein A/G–agarose, washed, incubated with 2 μ m sphingosine or left untreated, washed again, and incubated with the recombinant receptor-binding domain of the spike protein. The samples were washed extensively, eluted, separated by 7.5% SDS-PAGE electrophoresis, blotted, and developed with anti-spike antibodies. The recombinant receptor-binding domain of the spike protein is His-tagged and has a molecular mass of ∼27 kDa. Protein A/G–agarose beads without Fc-ACE2 served as control. Shown are representative results from five independent experiments. E , a variety of other lipids were added as indicated to immobilized recombinant human Fc-ACE2. The effects of these lipids on binding of recombinant receptor-binding domain of the spike protein were determined by Western blotting. Shown is a representative result from four independent studies. F , recombinant His-tagged RBD of spike was immobilized on Ni 2+ –agarose, washed, incubated with 2 μ m sphingosine, or left untreated. Recombinant human Fc-ACE2 was added, and the samples were incubated for 60 min. Beads without the addition of RBD spike served as control. The samples were washed extensively, eluted, separated by 7.5% SDS-PAGE electrophoresis, blotted, and developed with anti-ACE2 antibodies. Shown are representative results from five independent experiments. G , a variety of other lipids were added as indicated to immobilized recombinant RBD–spike protein. The effects of these lipids on binding of recombinant human Fc-ACE2 were determined by Western blotting. Shown is a representative result from four independent studies. PC , phosphatidylcholine; PE , <t>phosphatidylethanolamine;</t> SM , sphingomyelin; CER , ceramide; S1P , sphingosine 1-phosphate; Clp , cardiolipin; PS , phosphatidylserine; C16-CER , C16-ceramide; LC , lactosylceramide; OGP , octylglucopyranoside.
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    Echelon Biosciences phosphatidylcholine
    Sphingosine binds to ACE2 and thereby blocks the interaction of ACE2 with the viral spike protein. A , sphingosine (SPH)-coated beads or control ( Ctr ) beads were incubated with recombinant ( rec. ) Fc-ACE2 protein ( left panel ) or with lysates obtained from Vero cells ( right panel ), extensively washed, and eluted in 1× SDS sample buffer. As indicated, suspended sphingosine ( SPH ) was added (2 μ m ) to prevent binding of ACE2 to immobilized sphingosine. The samples were separated by 7.5% SDS-PAGE electrophoresis and blotted with anti-ACE2 antibodies. The Fc-ACE2 protein has a molecular mass of ∼180 kDa, and the endogenous ACE2 protein has a molecular mass of ∼120 kDa. Shown are representative results from five independent experiments. B , recombinant Fc-ACE2 protein was immobilized on protein A/G–agarose, washed, and incubated with 2 μ m sphingosine. Controls consisted of protein A/G–agarose only, incubated with 2 μ m sphingosine. The samples were washed and extracted in CHCl 3 :CH 3 OH:1N HCl (100:100:1, v/v/v), and sphingosine was quantified employing a kinase assay. Given are the means ± S.D. of the sphingosine concentrations from six independent experiments. ***, p < 0.001, ANOVA followed by post hoc Student's t tests. C , a panel of lipid-coated beads was used to test for the specificity of ACE2-binding to sphingosine-coupled beads. The samples were prepared as above. Shown is a representative result from four independent experiments. D , recombinant Fc-ACE2 protein was immobilized on protein A/G–agarose, washed, incubated with 2 μ m sphingosine or left untreated, washed again, and incubated with the recombinant receptor-binding domain of the spike protein. The samples were washed extensively, eluted, separated by 7.5% SDS-PAGE electrophoresis, blotted, and developed with anti-spike antibodies. The recombinant receptor-binding domain of the spike protein is His-tagged and has a molecular mass of ∼27 kDa. Protein A/G–agarose beads without Fc-ACE2 served as control. Shown are representative results from five independent experiments. E , a variety of other lipids were added as indicated to immobilized recombinant human Fc-ACE2. The effects of these lipids on binding of recombinant receptor-binding domain of the spike protein were determined by Western blotting. Shown is a representative result from four independent studies. F , recombinant His-tagged RBD of spike was immobilized on Ni 2+ –agarose, washed, incubated with 2 μ m sphingosine, or left untreated. Recombinant human Fc-ACE2 was added, and the samples were incubated for 60 min. Beads without the addition of RBD spike served as control. The samples were washed extensively, eluted, separated by 7.5% SDS-PAGE electrophoresis, blotted, and developed with anti-ACE2 antibodies. Shown are representative results from five independent experiments. G , a variety of other lipids were added as indicated to immobilized recombinant RBD–spike protein. The effects of these lipids on binding of recombinant human Fc-ACE2 were determined by Western blotting. Shown is a representative result from four independent studies. PC , phosphatidylcholine; PE , <t>phosphatidylethanolamine;</t> SM , sphingomyelin; CER , ceramide; S1P , sphingosine 1-phosphate; Clp , cardiolipin; PS , phosphatidylserine; C16-CER , C16-ceramide; LC , lactosylceramide; OGP , octylglucopyranoside.
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    Echelon Biosciences agarose sphingosine
    Sphingosine binds to ACE2 and thereby blocks the interaction of ACE2 with the viral spike protein. A , sphingosine (SPH)-coated beads or control ( Ctr ) beads were incubated with recombinant ( rec. ) Fc-ACE2 protein ( left panel ) or with lysates obtained from Vero cells ( right panel ), extensively washed, and eluted in 1× SDS sample buffer. As indicated, suspended sphingosine ( SPH ) was added (2 μ m ) to prevent binding of ACE2 to immobilized sphingosine. The samples were separated by 7.5% SDS-PAGE electrophoresis and blotted with anti-ACE2 antibodies. The Fc-ACE2 protein has a molecular mass of ∼180 kDa, and the endogenous ACE2 protein has a molecular mass of ∼120 kDa. Shown are representative results from five independent experiments. B , recombinant Fc-ACE2 protein was immobilized on protein A/G–agarose, washed, and incubated with 2 μ m sphingosine. Controls consisted of protein A/G–agarose only, incubated with 2 μ m sphingosine. The samples were washed and extracted in CHCl 3 :CH 3 OH:1N HCl (100:100:1, v/v/v), and sphingosine was quantified employing a kinase assay. Given are the means ± S.D. of the sphingosine concentrations from six independent experiments. ***, p < 0.001, ANOVA followed by post hoc Student's t tests. C , a panel of lipid-coated beads was used to test for the specificity of ACE2-binding to sphingosine-coupled beads. The samples were prepared as above. Shown is a representative result from four independent experiments. D , recombinant Fc-ACE2 protein was immobilized on protein A/G–agarose, washed, incubated with 2 μ m sphingosine or left untreated, washed again, and incubated with the recombinant receptor-binding domain of the spike protein. The samples were washed extensively, eluted, separated by 7.5% SDS-PAGE electrophoresis, blotted, and developed with anti-spike antibodies. The recombinant receptor-binding domain of the spike protein is His-tagged and has a molecular mass of ∼27 kDa. Protein A/G–agarose beads without Fc-ACE2 served as control. Shown are representative results from five independent experiments. E , a variety of other lipids were added as indicated to immobilized recombinant human Fc-ACE2. The effects of these lipids on binding of recombinant receptor-binding domain of the spike protein were determined by Western blotting. Shown is a representative result from four independent studies. F , recombinant His-tagged RBD of spike was immobilized on Ni 2+ –agarose, washed, incubated with 2 μ m sphingosine, or left untreated. Recombinant human Fc-ACE2 was added, and the samples were incubated for 60 min. Beads without the addition of RBD spike served as control. The samples were washed extensively, eluted, separated by 7.5% SDS-PAGE electrophoresis, blotted, and developed with anti-ACE2 antibodies. Shown are representative results from five independent experiments. G , a variety of other lipids were added as indicated to immobilized recombinant RBD–spike protein. The effects of these lipids on binding of recombinant human Fc-ACE2 were determined by Western blotting. Shown is a representative result from four independent studies. PC , phosphatidylcholine; PE , <t>phosphatidylethanolamine;</t> SM , sphingomyelin; CER , ceramide; S1P , sphingosine 1-phosphate; Clp , cardiolipin; PS , phosphatidylserine; C16-CER , C16-ceramide; LC , lactosylceramide; OGP , octylglucopyranoside.
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    Echelon Biosciences sphingosine
    Sphingosine binds to ACE2 and thereby blocks the interaction of ACE2 with the viral spike protein. A , sphingosine (SPH)-coated beads or control ( Ctr ) beads were incubated with recombinant ( rec. ) Fc-ACE2 protein ( left panel ) or with lysates obtained from Vero cells ( right panel ), extensively washed, and eluted in 1× SDS sample buffer. As indicated, suspended sphingosine ( SPH ) was added (2 μ m ) to prevent binding of ACE2 to immobilized sphingosine. The samples were separated by 7.5% SDS-PAGE electrophoresis and blotted with anti-ACE2 antibodies. The Fc-ACE2 protein has a molecular mass of ∼180 kDa, and the endogenous ACE2 protein has a molecular mass of ∼120 kDa. Shown are representative results from five independent experiments. B , recombinant Fc-ACE2 protein was immobilized on protein A/G–agarose, washed, and incubated with 2 μ m sphingosine. Controls consisted of protein A/G–agarose only, incubated with 2 μ m sphingosine. The samples were washed and extracted in CHCl 3 :CH 3 OH:1N HCl (100:100:1, v/v/v), and sphingosine was quantified employing a kinase assay. Given are the means ± S.D. of the sphingosine concentrations from six independent experiments. ***, p < 0.001, ANOVA followed by post hoc Student's t tests. C , a panel of lipid-coated beads was used to test for the specificity of ACE2-binding to sphingosine-coupled beads. The samples were prepared as above. Shown is a representative result from four independent experiments. D , recombinant Fc-ACE2 protein was immobilized on protein A/G–agarose, washed, incubated with 2 μ m sphingosine or left untreated, washed again, and incubated with the recombinant receptor-binding domain of the spike protein. The samples were washed extensively, eluted, separated by 7.5% SDS-PAGE electrophoresis, blotted, and developed with anti-spike antibodies. The recombinant receptor-binding domain of the spike protein is His-tagged and has a molecular mass of ∼27 kDa. Protein A/G–agarose beads without Fc-ACE2 served as control. Shown are representative results from five independent experiments. E , a variety of other lipids were added as indicated to immobilized recombinant human Fc-ACE2. The effects of these lipids on binding of recombinant receptor-binding domain of the spike protein were determined by Western blotting. Shown is a representative result from four independent studies. F , recombinant His-tagged RBD of spike was immobilized on Ni 2+ –agarose, washed, incubated with 2 μ m sphingosine, or left untreated. Recombinant human Fc-ACE2 was added, and the samples were incubated for 60 min. Beads without the addition of RBD spike served as control. The samples were washed extensively, eluted, separated by 7.5% SDS-PAGE electrophoresis, blotted, and developed with anti-ACE2 antibodies. Shown are representative results from five independent experiments. G , a variety of other lipids were added as indicated to immobilized recombinant RBD–spike protein. The effects of these lipids on binding of recombinant human Fc-ACE2 were determined by Western blotting. Shown is a representative result from four independent studies. PC , phosphatidylcholine; PE , <t>phosphatidylethanolamine;</t> SM , sphingomyelin; CER , ceramide; S1P , sphingosine 1-phosphate; Clp , cardiolipin; PS , phosphatidylserine; C16-CER , C16-ceramide; LC , lactosylceramide; OGP , octylglucopyranoside.
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    Sphingosine binds to ACE2 and thereby blocks the interaction of ACE2 with the viral spike protein. A , sphingosine (SPH)-coated beads or control ( Ctr ) beads were incubated with recombinant ( rec. ) Fc-ACE2 protein ( left panel ) or with lysates obtained from Vero cells ( right panel ), extensively washed, and eluted in 1× SDS sample buffer. As indicated, suspended sphingosine ( SPH ) was added (2 μ m ) to prevent binding of ACE2 to immobilized sphingosine. The samples were separated by 7.5% SDS-PAGE electrophoresis and blotted with anti-ACE2 antibodies. The Fc-ACE2 protein has a molecular mass of ∼180 kDa, and the endogenous ACE2 protein has a molecular mass of ∼120 kDa. Shown are representative results from five independent experiments. B , recombinant Fc-ACE2 protein was immobilized on protein A/G–agarose, washed, and incubated with 2 μ m sphingosine. Controls consisted of protein A/G–agarose only, incubated with 2 μ m sphingosine. The samples were washed and extracted in CHCl 3 :CH 3 OH:1N HCl (100:100:1, v/v/v), and sphingosine was quantified employing a kinase assay. Given are the means ± S.D. of the sphingosine concentrations from six independent experiments. ***, p < 0.001, ANOVA followed by post hoc Student's t tests. C , a panel of lipid-coated beads was used to test for the specificity of ACE2-binding to sphingosine-coupled beads. The samples were prepared as above. Shown is a representative result from four independent experiments. D , recombinant Fc-ACE2 protein was immobilized on protein A/G–agarose, washed, incubated with 2 μ m sphingosine or left untreated, washed again, and incubated with the recombinant receptor-binding domain of the spike protein. The samples were washed extensively, eluted, separated by 7.5% SDS-PAGE electrophoresis, blotted, and developed with anti-spike antibodies. The recombinant receptor-binding domain of the spike protein is His-tagged and has a molecular mass of ∼27 kDa. Protein A/G–agarose beads without Fc-ACE2 served as control. Shown are representative results from five independent experiments. E , a variety of other lipids were added as indicated to immobilized recombinant human Fc-ACE2. The effects of these lipids on binding of recombinant receptor-binding domain of the spike protein were determined by Western blotting. Shown is a representative result from four independent studies. F , recombinant His-tagged RBD of spike was immobilized on Ni 2+ –agarose, washed, incubated with 2 μ m sphingosine, or left untreated. Recombinant human Fc-ACE2 was added, and the samples were incubated for 60 min. Beads without the addition of RBD spike served as control. The samples were washed extensively, eluted, separated by 7.5% SDS-PAGE electrophoresis, blotted, and developed with anti-ACE2 antibodies. Shown are representative results from five independent experiments. G , a variety of other lipids were added as indicated to immobilized recombinant RBD–spike protein. The effects of these lipids on binding of recombinant human Fc-ACE2 were determined by Western blotting. Shown is a representative result from four independent studies. PC , phosphatidylcholine; PE , phosphatidylethanolamine; SM , sphingomyelin; CER , ceramide; S1P , sphingosine 1-phosphate; Clp , cardiolipin; PS , phosphatidylserine; C16-CER , C16-ceramide; LC , lactosylceramide; OGP , octylglucopyranoside.

    Journal: The Journal of Biological Chemistry

    Article Title: Sphingosine prevents binding of SARS–CoV-2 spike to its cellular receptor ACE2

    doi: 10.1074/jbc.RA120.015249

    Figure Lengend Snippet: Sphingosine binds to ACE2 and thereby blocks the interaction of ACE2 with the viral spike protein. A , sphingosine (SPH)-coated beads or control ( Ctr ) beads were incubated with recombinant ( rec. ) Fc-ACE2 protein ( left panel ) or with lysates obtained from Vero cells ( right panel ), extensively washed, and eluted in 1× SDS sample buffer. As indicated, suspended sphingosine ( SPH ) was added (2 μ m ) to prevent binding of ACE2 to immobilized sphingosine. The samples were separated by 7.5% SDS-PAGE electrophoresis and blotted with anti-ACE2 antibodies. The Fc-ACE2 protein has a molecular mass of ∼180 kDa, and the endogenous ACE2 protein has a molecular mass of ∼120 kDa. Shown are representative results from five independent experiments. B , recombinant Fc-ACE2 protein was immobilized on protein A/G–agarose, washed, and incubated with 2 μ m sphingosine. Controls consisted of protein A/G–agarose only, incubated with 2 μ m sphingosine. The samples were washed and extracted in CHCl 3 :CH 3 OH:1N HCl (100:100:1, v/v/v), and sphingosine was quantified employing a kinase assay. Given are the means ± S.D. of the sphingosine concentrations from six independent experiments. ***, p < 0.001, ANOVA followed by post hoc Student's t tests. C , a panel of lipid-coated beads was used to test for the specificity of ACE2-binding to sphingosine-coupled beads. The samples were prepared as above. Shown is a representative result from four independent experiments. D , recombinant Fc-ACE2 protein was immobilized on protein A/G–agarose, washed, incubated with 2 μ m sphingosine or left untreated, washed again, and incubated with the recombinant receptor-binding domain of the spike protein. The samples were washed extensively, eluted, separated by 7.5% SDS-PAGE electrophoresis, blotted, and developed with anti-spike antibodies. The recombinant receptor-binding domain of the spike protein is His-tagged and has a molecular mass of ∼27 kDa. Protein A/G–agarose beads without Fc-ACE2 served as control. Shown are representative results from five independent experiments. E , a variety of other lipids were added as indicated to immobilized recombinant human Fc-ACE2. The effects of these lipids on binding of recombinant receptor-binding domain of the spike protein were determined by Western blotting. Shown is a representative result from four independent studies. F , recombinant His-tagged RBD of spike was immobilized on Ni 2+ –agarose, washed, incubated with 2 μ m sphingosine, or left untreated. Recombinant human Fc-ACE2 was added, and the samples were incubated for 60 min. Beads without the addition of RBD spike served as control. The samples were washed extensively, eluted, separated by 7.5% SDS-PAGE electrophoresis, blotted, and developed with anti-ACE2 antibodies. Shown are representative results from five independent experiments. G , a variety of other lipids were added as indicated to immobilized recombinant RBD–spike protein. The effects of these lipids on binding of recombinant human Fc-ACE2 were determined by Western blotting. Shown is a representative result from four independent studies. PC , phosphatidylcholine; PE , phosphatidylethanolamine; SM , sphingomyelin; CER , ceramide; S1P , sphingosine 1-phosphate; Clp , cardiolipin; PS , phosphatidylserine; C16-CER , C16-ceramide; LC , lactosylceramide; OGP , octylglucopyranoside.

    Article Snippet: The following lipids were immobilized to agarose: sphingosine (Sphingobeads, Echelon, catalog no. S-6100), phosphatidylcholine (Echelon, catalog no. P-BOPC), phosphatidylethanolamine (Echelon, catalog no. P-BOPE), sphingomyelin (Echelon, catalog no. P-BOSM), ceramide (Echelon, catalog no. P-BCER), sphingosine 1-phosphate (Echelon, catalog no. S6110, cardiolipin (Echelon, catalog no. P-BCLP), or unloaded control beads (Echelon, catalog no. P-8000).

    Techniques: Incubation, Recombinant, Binding Assay, SDS Page, Electrophoresis, Kinase Assay, Western Blot