Structured Review

Roche mgcl
Development of hydrolysis resistant hSTING agonists ( a ) Scheme of enzymatic synthesis of 3′3′-cGAMP and 2′3′-cGAMP analogs. For 3′3′-cGAMP, 50 nM of DncV was incubated with 1 mM ATP and 1 mM GTP in 1 mL buffer containing 20 mM <t>Tris-HCl,</t> pH 8.0, 20 mM <t>MgCl</t> 2 for 3 h at room temperature. For 2′3′-cGAMP analogs, 1-10 μM mouse cGAS (amino acid residues 147-507) was incubated with 1 mM ATP, 1 mM GTP, and 0.1 mg/mL HT-DNA in 1 mL of the same buffer for 12 h at room temperature. ( b ) Hydrolysis reactions of ENPP1 (1 nM) with the analogs (10 μM). ( c ) Scintillation proximity assay to measure the binding affinity of the analogs towards hSTING (amino acid residues 139-379). Biotinylated hSTING (100 nM) was immobilized onto 96-well streptavidin coated SPA plates. Neat 35 S-labeled 2′3′-cGA s MP (500 pM) was used as the probe. N=3 samples. Data are presented as mean and standard error. ( d ) IFN-β production in THP-1 cells stimulated with the analogs. THP-1 cells were incubated with the analogs at the indicated concentrations for 24 h. IFN-β in the media was measured using a HEK-SEAP cell line. Representative data from a biological triplicate is depicted.
Mgcl, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Article Title: Hydrolysis of 2′3′-cGAMP by ENPP1 and design of non-hydrolyzable analogs

Journal: Nature chemical biology

doi: 10.1038/nchembio.1661

Development of hydrolysis resistant hSTING agonists ( a ) Scheme of enzymatic synthesis of 3′3′-cGAMP and 2′3′-cGAMP analogs. For 3′3′-cGAMP, 50 nM of DncV was incubated with 1 mM ATP and 1 mM GTP in 1 mL buffer containing 20 mM Tris-HCl, pH 8.0, 20 mM MgCl 2 for 3 h at room temperature. For 2′3′-cGAMP analogs, 1-10 μM mouse cGAS (amino acid residues 147-507) was incubated with 1 mM ATP, 1 mM GTP, and 0.1 mg/mL HT-DNA in 1 mL of the same buffer for 12 h at room temperature. ( b ) Hydrolysis reactions of ENPP1 (1 nM) with the analogs (10 μM). ( c ) Scintillation proximity assay to measure the binding affinity of the analogs towards hSTING (amino acid residues 139-379). Biotinylated hSTING (100 nM) was immobilized onto 96-well streptavidin coated SPA plates. Neat 35 S-labeled 2′3′-cGA s MP (500 pM) was used as the probe. N=3 samples. Data are presented as mean and standard error. ( d ) IFN-β production in THP-1 cells stimulated with the analogs. THP-1 cells were incubated with the analogs at the indicated concentrations for 24 h. IFN-β in the media was measured using a HEK-SEAP cell line. Representative data from a biological triplicate is depicted.
Figure Legend Snippet: Development of hydrolysis resistant hSTING agonists ( a ) Scheme of enzymatic synthesis of 3′3′-cGAMP and 2′3′-cGAMP analogs. For 3′3′-cGAMP, 50 nM of DncV was incubated with 1 mM ATP and 1 mM GTP in 1 mL buffer containing 20 mM Tris-HCl, pH 8.0, 20 mM MgCl 2 for 3 h at room temperature. For 2′3′-cGAMP analogs, 1-10 μM mouse cGAS (amino acid residues 147-507) was incubated with 1 mM ATP, 1 mM GTP, and 0.1 mg/mL HT-DNA in 1 mL of the same buffer for 12 h at room temperature. ( b ) Hydrolysis reactions of ENPP1 (1 nM) with the analogs (10 μM). ( c ) Scintillation proximity assay to measure the binding affinity of the analogs towards hSTING (amino acid residues 139-379). Biotinylated hSTING (100 nM) was immobilized onto 96-well streptavidin coated SPA plates. Neat 35 S-labeled 2′3′-cGA s MP (500 pM) was used as the probe. N=3 samples. Data are presented as mean and standard error. ( d ) IFN-β production in THP-1 cells stimulated with the analogs. THP-1 cells were incubated with the analogs at the indicated concentrations for 24 h. IFN-β in the media was measured using a HEK-SEAP cell line. Representative data from a biological triplicate is depicted.

Techniques Used: Incubation, Scintillation Proximity Assay, Binding Assay, Labeling

Related Articles

Multiple Displacement Amplification:

Article Title: Hydrolysis of 2′3′-cGAMP by ENPP1 and design of non-hydrolyzable analogs
Article Snippet: .. Preparation of cell and tissue extracts To determine whether THP-1 cells, L929 cells, and MDA-MB231 possess hydrolase activity for 2′3′-cGAMP, the whole cell lysate was generated using lysis buffer:10 mM Tris-HCl, pH 7.5,150 mM NaCl, 1% NP-40, 1.5 mM MgCl, 2 mM DTT, protease inhibitor tablet (Roche) at 1 mL for 100 million cells. .. To determine the subcellular location of the hydrolase(s), cells were lysed by douncing in either a mitochondria friendly buffer: 10 mM Tris-MOPS (pH 7.4), 10 mM EDTA/Tris, 200 mM sucrose or a hypotonic buffer: 10 mM Tris-HCl, pH 7.5, 10 mM KCl, 1.5 mM MgCl2 , 2 mM DTT, protease inhibitor tablet.

Protease Inhibitor:

Article Title: Hydrolysis of 2′3′-cGAMP by ENPP1 and design of non-hydrolyzable analogs
Article Snippet: .. Preparation of cell and tissue extracts To determine whether THP-1 cells, L929 cells, and MDA-MB231 possess hydrolase activity for 2′3′-cGAMP, the whole cell lysate was generated using lysis buffer:10 mM Tris-HCl, pH 7.5,150 mM NaCl, 1% NP-40, 1.5 mM MgCl, 2 mM DTT, protease inhibitor tablet (Roche) at 1 mL for 100 million cells. .. To determine the subcellular location of the hydrolase(s), cells were lysed by douncing in either a mitochondria friendly buffer: 10 mM Tris-MOPS (pH 7.4), 10 mM EDTA/Tris, 200 mM sucrose or a hypotonic buffer: 10 mM Tris-HCl, pH 7.5, 10 mM KCl, 1.5 mM MgCl2 , 2 mM DTT, protease inhibitor tablet.

Article Title: Lysine Deacetylases Hda1 and Rpd3 Regulate Hsp90 Function thereby Governing Fungal Drug Resistance
Article Snippet: .. In brief, cells were washed with sterile water and resuspended in 500 μl of lysis buffer J containing 20 mM Tris pH 7.5, 100 mM KCl, 5 mM MgCl and 20% glycerol, with one protease inhibitor cocktail (complete, EDTA-free tablet, Roche Diagnostics) per 10 mL, as well as 1 mM PMSF (EMD Chemicals) and 20 mM sodium molybdate (Sigma Aldrich Co.) added fresh before use. ..

Article Title: The Nucleosome Assembly Activity of NAP1 Is Enhanced by Alien ▿
Article Snippet: .. Bacterially expressed GST, GST-Alien, and deletions were bound to glutathione-Sepharose beads and blocked with BSA before incubation with 10 μg of core histones (Roche) in the presence of BSA for 1 h. Beads were washed extensively with a buffer containing 20 mM Tris-HCl, pH 7.5, 270 mM KCl, 5 mM MgCl, 1% NP-40, 1 mM dithiothreitol, 10 μg/ml BSA, and protease inhibitor cocktail complete (Roche). .. ChIP and re-ChIP were performed essentially as described earlier ( ).

Generated:

Article Title: Hydrolysis of 2′3′-cGAMP by ENPP1 and design of non-hydrolyzable analogs
Article Snippet: .. Preparation of cell and tissue extracts To determine whether THP-1 cells, L929 cells, and MDA-MB231 possess hydrolase activity for 2′3′-cGAMP, the whole cell lysate was generated using lysis buffer:10 mM Tris-HCl, pH 7.5,150 mM NaCl, 1% NP-40, 1.5 mM MgCl, 2 mM DTT, protease inhibitor tablet (Roche) at 1 mL for 100 million cells. .. To determine the subcellular location of the hydrolase(s), cells were lysed by douncing in either a mitochondria friendly buffer: 10 mM Tris-MOPS (pH 7.4), 10 mM EDTA/Tris, 200 mM sucrose or a hypotonic buffer: 10 mM Tris-HCl, pH 7.5, 10 mM KCl, 1.5 mM MgCl2 , 2 mM DTT, protease inhibitor tablet.

Incubation:

Article Title: The Nucleosome Assembly Activity of NAP1 Is Enhanced by Alien ▿
Article Snippet: .. Bacterially expressed GST, GST-Alien, and deletions were bound to glutathione-Sepharose beads and blocked with BSA before incubation with 10 μg of core histones (Roche) in the presence of BSA for 1 h. Beads were washed extensively with a buffer containing 20 mM Tris-HCl, pH 7.5, 270 mM KCl, 5 mM MgCl, 1% NP-40, 1 mM dithiothreitol, 10 μg/ml BSA, and protease inhibitor cocktail complete (Roche). .. ChIP and re-ChIP were performed essentially as described earlier ( ).

Activity Assay:

Article Title: Hydrolysis of 2′3′-cGAMP by ENPP1 and design of non-hydrolyzable analogs
Article Snippet: .. Preparation of cell and tissue extracts To determine whether THP-1 cells, L929 cells, and MDA-MB231 possess hydrolase activity for 2′3′-cGAMP, the whole cell lysate was generated using lysis buffer:10 mM Tris-HCl, pH 7.5,150 mM NaCl, 1% NP-40, 1.5 mM MgCl, 2 mM DTT, protease inhibitor tablet (Roche) at 1 mL for 100 million cells. .. To determine the subcellular location of the hydrolase(s), cells were lysed by douncing in either a mitochondria friendly buffer: 10 mM Tris-MOPS (pH 7.4), 10 mM EDTA/Tris, 200 mM sucrose or a hypotonic buffer: 10 mM Tris-HCl, pH 7.5, 10 mM KCl, 1.5 mM MgCl2 , 2 mM DTT, protease inhibitor tablet.

Release Assay:

Article Title: Betulinic Acid Selectively Increases Protein Degradation and Enhances Prostate Cancer-Specific Apoptosis: Possible Role for Inhibition of Deubiquitinase Activity
Article Snippet: .. Mitochondrial protein release assay Treated and control PC cells were resuspended in a buffer containing 100–200 µM digitonin, 20 mM Hepes, pH 7.5, 10 mM KCl, 1.5 mM MgCl, 1 mM EGTA, 1 mM EDTA, 1 mM DTT, 250 mM sucrose, and protease inhibitors (Roche) at 50 µl/1×106 cells. ..

Lysis:

Article Title: Hydrolysis of 2′3′-cGAMP by ENPP1 and design of non-hydrolyzable analogs
Article Snippet: .. Preparation of cell and tissue extracts To determine whether THP-1 cells, L929 cells, and MDA-MB231 possess hydrolase activity for 2′3′-cGAMP, the whole cell lysate was generated using lysis buffer:10 mM Tris-HCl, pH 7.5,150 mM NaCl, 1% NP-40, 1.5 mM MgCl, 2 mM DTT, protease inhibitor tablet (Roche) at 1 mL for 100 million cells. .. To determine the subcellular location of the hydrolase(s), cells were lysed by douncing in either a mitochondria friendly buffer: 10 mM Tris-MOPS (pH 7.4), 10 mM EDTA/Tris, 200 mM sucrose or a hypotonic buffer: 10 mM Tris-HCl, pH 7.5, 10 mM KCl, 1.5 mM MgCl2 , 2 mM DTT, protease inhibitor tablet.

Article Title: Lysine Deacetylases Hda1 and Rpd3 Regulate Hsp90 Function thereby Governing Fungal Drug Resistance
Article Snippet: .. In brief, cells were washed with sterile water and resuspended in 500 μl of lysis buffer J containing 20 mM Tris pH 7.5, 100 mM KCl, 5 mM MgCl and 20% glycerol, with one protease inhibitor cocktail (complete, EDTA-free tablet, Roche Diagnostics) per 10 mL, as well as 1 mM PMSF (EMD Chemicals) and 20 mM sodium molybdate (Sigma Aldrich Co.) added fresh before use. ..

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    Roche mgcl
    Development of hydrolysis resistant hSTING agonists ( a ) Scheme of enzymatic synthesis of 3′3′-cGAMP and 2′3′-cGAMP analogs. For 3′3′-cGAMP, 50 nM of DncV was incubated with 1 mM ATP and 1 mM GTP in 1 mL buffer containing 20 mM <t>Tris-HCl,</t> pH 8.0, 20 mM <t>MgCl</t> 2 for 3 h at room temperature. For 2′3′-cGAMP analogs, 1-10 μM mouse cGAS (amino acid residues 147-507) was incubated with 1 mM ATP, 1 mM GTP, and 0.1 mg/mL HT-DNA in 1 mL of the same buffer for 12 h at room temperature. ( b ) Hydrolysis reactions of ENPP1 (1 nM) with the analogs (10 μM). ( c ) Scintillation proximity assay to measure the binding affinity of the analogs towards hSTING (amino acid residues 139-379). Biotinylated hSTING (100 nM) was immobilized onto 96-well streptavidin coated SPA plates. Neat 35 S-labeled 2′3′-cGA s MP (500 pM) was used as the probe. N=3 samples. Data are presented as mean and standard error. ( d ) IFN-β production in THP-1 cells stimulated with the analogs. THP-1 cells were incubated with the analogs at the indicated concentrations for 24 h. IFN-β in the media was measured using a HEK-SEAP cell line. Representative data from a biological triplicate is depicted.
    Mgcl, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mgcl/product/Roche
    Average 93 stars, based on 19 article reviews
    Price from $9.99 to $1999.99
    mgcl - by Bioz Stars, 2020-07
    93/100 stars
      Buy from Supplier

    85
    Roche tris hcl mgcl reaction buffer
    Development of hydrolysis resistant hSTING agonists ( a ) Scheme of enzymatic synthesis of 3′3′-cGAMP and 2′3′-cGAMP analogs. For 3′3′-cGAMP, 50 nM of DncV was incubated with 1 mM ATP and 1 mM GTP in 1 mL buffer containing 20 mM <t>Tris-HCl,</t> pH 8.0, 20 mM <t>MgCl</t> 2 for 3 h at room temperature. For 2′3′-cGAMP analogs, 1-10 μM mouse cGAS (amino acid residues 147-507) was incubated with 1 mM ATP, 1 mM GTP, and 0.1 mg/mL HT-DNA in 1 mL of the same buffer for 12 h at room temperature. ( b ) Hydrolysis reactions of ENPP1 (1 nM) with the analogs (10 μM). ( c ) Scintillation proximity assay to measure the binding affinity of the analogs towards hSTING (amino acid residues 139-379). Biotinylated hSTING (100 nM) was immobilized onto 96-well streptavidin coated SPA plates. Neat 35 S-labeled 2′3′-cGA s MP (500 pM) was used as the probe. N=3 samples. Data are presented as mean and standard error. ( d ) IFN-β production in THP-1 cells stimulated with the analogs. THP-1 cells were incubated with the analogs at the indicated concentrations for 24 h. IFN-β in the media was measured using a HEK-SEAP cell line. Representative data from a biological triplicate is depicted.
    Tris Hcl Mgcl Reaction Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Development of hydrolysis resistant hSTING agonists ( a ) Scheme of enzymatic synthesis of 3′3′-cGAMP and 2′3′-cGAMP analogs. For 3′3′-cGAMP, 50 nM of DncV was incubated with 1 mM ATP and 1 mM GTP in 1 mL buffer containing 20 mM Tris-HCl, pH 8.0, 20 mM MgCl 2 for 3 h at room temperature. For 2′3′-cGAMP analogs, 1-10 μM mouse cGAS (amino acid residues 147-507) was incubated with 1 mM ATP, 1 mM GTP, and 0.1 mg/mL HT-DNA in 1 mL of the same buffer for 12 h at room temperature. ( b ) Hydrolysis reactions of ENPP1 (1 nM) with the analogs (10 μM). ( c ) Scintillation proximity assay to measure the binding affinity of the analogs towards hSTING (amino acid residues 139-379). Biotinylated hSTING (100 nM) was immobilized onto 96-well streptavidin coated SPA plates. Neat 35 S-labeled 2′3′-cGA s MP (500 pM) was used as the probe. N=3 samples. Data are presented as mean and standard error. ( d ) IFN-β production in THP-1 cells stimulated with the analogs. THP-1 cells were incubated with the analogs at the indicated concentrations for 24 h. IFN-β in the media was measured using a HEK-SEAP cell line. Representative data from a biological triplicate is depicted.

    Journal: Nature chemical biology

    Article Title: Hydrolysis of 2′3′-cGAMP by ENPP1 and design of non-hydrolyzable analogs

    doi: 10.1038/nchembio.1661

    Figure Lengend Snippet: Development of hydrolysis resistant hSTING agonists ( a ) Scheme of enzymatic synthesis of 3′3′-cGAMP and 2′3′-cGAMP analogs. For 3′3′-cGAMP, 50 nM of DncV was incubated with 1 mM ATP and 1 mM GTP in 1 mL buffer containing 20 mM Tris-HCl, pH 8.0, 20 mM MgCl 2 for 3 h at room temperature. For 2′3′-cGAMP analogs, 1-10 μM mouse cGAS (amino acid residues 147-507) was incubated with 1 mM ATP, 1 mM GTP, and 0.1 mg/mL HT-DNA in 1 mL of the same buffer for 12 h at room temperature. ( b ) Hydrolysis reactions of ENPP1 (1 nM) with the analogs (10 μM). ( c ) Scintillation proximity assay to measure the binding affinity of the analogs towards hSTING (amino acid residues 139-379). Biotinylated hSTING (100 nM) was immobilized onto 96-well streptavidin coated SPA plates. Neat 35 S-labeled 2′3′-cGA s MP (500 pM) was used as the probe. N=3 samples. Data are presented as mean and standard error. ( d ) IFN-β production in THP-1 cells stimulated with the analogs. THP-1 cells were incubated with the analogs at the indicated concentrations for 24 h. IFN-β in the media was measured using a HEK-SEAP cell line. Representative data from a biological triplicate is depicted.

    Article Snippet: Preparation of cell and tissue extracts To determine whether THP-1 cells, L929 cells, and MDA-MB231 possess hydrolase activity for 2′3′-cGAMP, the whole cell lysate was generated using lysis buffer:10 mM Tris-HCl, pH 7.5,150 mM NaCl, 1% NP-40, 1.5 mM MgCl, 2 mM DTT, protease inhibitor tablet (Roche) at 1 mL for 100 million cells.

    Techniques: Incubation, Scintillation Proximity Assay, Binding Assay, Labeling