protease inhibitor mixture  (Promega)

 
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  • 99
    Name:
    Protease Inhibitor Cocktail
    Description:
    Prevents protein degradation after cell lysis For protein isolation from mammalian or insect cell cultures
    Catalog Number:
    g6521
    Price:
    None
    Category:
    Protein Analysis Protein Interactions Pull Down and Two Hybrid Systems
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    Structured Review

    Promega protease inhibitor mixture
    Prevents protein degradation after cell lysis For protein isolation from mammalian or insect cell cultures
    https://www.bioz.com/result/protease inhibitor mixture/product/Promega
    Average 99 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    protease inhibitor mixture - by Bioz Stars, 2020-09
    99/100 stars

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    Related Articles

    Transfection:

    Article Title: miR-512-5p suppresses proliferation, migration and invasion, and induces apoptosis in non-small cell lung cancer cells by targeting ETS1
    Article Snippet: .. Western blot analysis Following transfection, cells of 80% density were lysed using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Haimen, China) containing a Protease Inhibitor Cocktail (Promega Corporation) for 30 min. .. The protein was collected, and the concentrations were measured using a BCA assay kit (Beyotime Institute of Biotechnology).

    In Vitro:

    Article Title: Inhibition of Japanese encephalitis virus infection by the host zinc-finger antiviral protein
    Article Snippet: .. In vitro RNA pull-down assay Different amounts (0, 1, 5, and 10 pmole) of biotin-labeled RNA probe were incubated with 100 μg of ZAP-S-V5 or ZAP-S-del4ZFs expressing A549 cell extracts lysed by CHAPS lysis buffer (10 mM Tris-HCl [pH 7.4], 1 mM MgCl2 , 1 mM EGTA, 0.5% CHAPS, 10% glycerol, and 5 mM 2-mercaptoethanol) containing protease inhibitor cocktail in a final volume of 100 μl of RNA binding buffer (10 mM Tris-HCl [pH 7.5], 50 mM NaCl, 1 mM EDTA, and 10 μM ZnCl2 ) supplemented with 1 unit/μl RNasin (Promega), 1 μg/μl heparin (Sigma-Aldrich) and 100 ng/μl yeast tRNA (Roche) for 30 min at 30°C. .. After incubation, 300 μl of Streptavidin MagneSphere paramagnetic particles (Promega) was added for 30 min at 25°C.

    Radio Immunoprecipitation:

    Article Title: miR-512-5p suppresses proliferation, migration and invasion, and induces apoptosis in non-small cell lung cancer cells by targeting ETS1
    Article Snippet: .. Western blot analysis Following transfection, cells of 80% density were lysed using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Haimen, China) containing a Protease Inhibitor Cocktail (Promega Corporation) for 30 min. .. The protein was collected, and the concentrations were measured using a BCA assay kit (Beyotime Institute of Biotechnology).

    Article Title: Systemic Delivery of Gemcitabine Triphosphate via LCP Nanoparticles for NSCLC and Pancreatic Cancer Therapy
    Article Snippet: .. The cells and tumor tissues were lysed with a radioimmunoprecipitation assay (RIPA) buffer that was supplemented with a protease inhibitor cocktail (Promega, Madison, WI). ..

    Protease Inhibitor:

    Article Title: The Pro-Inflammatory Cytokine, Interleukin-6, Enhances the Polarization of Alternatively Activated Macrophages
    Article Snippet: .. Immunoblotting Briefly, cells were lysed in modified RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS) supplemented with protease inhibitor cocktail (Promega, Madison, WI) and protein concentrations determined via the Bradford assay (Bio-Rad Laboratories Canada, Mississauga, ON, Canada). .. Samples (10–20 µg) were boiled for 10 min with 4x Laemelli buffer, run by SDS-PAGE (4% stacking, 8% separating) and transferred to a nitrocellulose membrane.

    Article Title: Dopamine D1A directly interacts with otoferlin synaptic pathway proteins: Ca2+ and phosphorylation underlie an NSF-to-AP2mu1 molecular switch
    Article Snippet: .. For co-immunoprecipitation analyses, rat brain tissue was solubilized in calcium-free HBS buffer containing 0.1% Tween-20 and protease inhibitor cocktails (Promega) by brief sonication on ice. .. The supernatant from cold centrifugation at 20 900 × g for 12 min was diluted to a concentration of 1 mg/ml with calcium-free HBS buffer containing a protease inhibitor.

    Article Title: miR-512-5p suppresses proliferation, migration and invasion, and induces apoptosis in non-small cell lung cancer cells by targeting ETS1
    Article Snippet: .. Western blot analysis Following transfection, cells of 80% density were lysed using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Haimen, China) containing a Protease Inhibitor Cocktail (Promega Corporation) for 30 min. .. The protein was collected, and the concentrations were measured using a BCA assay kit (Beyotime Institute of Biotechnology).

    Article Title: Identifying Dysregulated Epigenetic Enzyme Activity in Castrate-Resistant Prostate Cancer Development
    Article Snippet: .. Before each assay, cell pellets from PCa cell lines (LAPC4, LNCaP, PC3, DU145, 22RV1, and C4–2) were thawed on ice and resuspended in 300 μ L of lysis buffer; 50 mM Tris-HCl at pH 7.5; 150 mM NaCl; 1% Triton X-100; DNase I (Roche); protease inhibitor cocktail (Promega); and NaF and incubated at RT for 10 min with gentle rotation. ..

    Article Title: Inhibition of Japanese encephalitis virus infection by the host zinc-finger antiviral protein
    Article Snippet: .. In vitro RNA pull-down assay Different amounts (0, 1, 5, and 10 pmole) of biotin-labeled RNA probe were incubated with 100 μg of ZAP-S-V5 or ZAP-S-del4ZFs expressing A549 cell extracts lysed by CHAPS lysis buffer (10 mM Tris-HCl [pH 7.4], 1 mM MgCl2 , 1 mM EGTA, 0.5% CHAPS, 10% glycerol, and 5 mM 2-mercaptoethanol) containing protease inhibitor cocktail in a final volume of 100 μl of RNA binding buffer (10 mM Tris-HCl [pH 7.5], 50 mM NaCl, 1 mM EDTA, and 10 μM ZnCl2 ) supplemented with 1 unit/μl RNasin (Promega), 1 μg/μl heparin (Sigma-Aldrich) and 100 ng/μl yeast tRNA (Roche) for 30 min at 30°C. .. After incubation, 300 μl of Streptavidin MagneSphere paramagnetic particles (Promega) was added for 30 min at 25°C.

    Article Title: ER stress-linked autophagy stabilizes apoptosis effector PERP and triggers its co-localization with SERCA2b at ER–plasma membrane junctions
    Article Snippet: .. The resulting pellet was lysed in Mammalian Lysis Buffer with Protease Inhibitor Cocktail (both from Promega) and 1.5 mg of protein was incubated with the bead–antibody conjugate for 30 min at room temperature. .. Antibody–protein complexes were washed four times in PBS and proteins were removed in SDS sample buffer for Western blot analysis.

    Article Title: Systemic Delivery of Gemcitabine Triphosphate via LCP Nanoparticles for NSCLC and Pancreatic Cancer Therapy
    Article Snippet: .. The cells and tumor tissues were lysed with a radioimmunoprecipitation assay (RIPA) buffer that was supplemented with a protease inhibitor cocktail (Promega, Madison, WI). ..

    Incubation:

    Article Title: Identifying Dysregulated Epigenetic Enzyme Activity in Castrate-Resistant Prostate Cancer Development
    Article Snippet: .. Before each assay, cell pellets from PCa cell lines (LAPC4, LNCaP, PC3, DU145, 22RV1, and C4–2) were thawed on ice and resuspended in 300 μ L of lysis buffer; 50 mM Tris-HCl at pH 7.5; 150 mM NaCl; 1% Triton X-100; DNase I (Roche); protease inhibitor cocktail (Promega); and NaF and incubated at RT for 10 min with gentle rotation. ..

    Article Title: Inhibition of Japanese encephalitis virus infection by the host zinc-finger antiviral protein
    Article Snippet: .. In vitro RNA pull-down assay Different amounts (0, 1, 5, and 10 pmole) of biotin-labeled RNA probe were incubated with 100 μg of ZAP-S-V5 or ZAP-S-del4ZFs expressing A549 cell extracts lysed by CHAPS lysis buffer (10 mM Tris-HCl [pH 7.4], 1 mM MgCl2 , 1 mM EGTA, 0.5% CHAPS, 10% glycerol, and 5 mM 2-mercaptoethanol) containing protease inhibitor cocktail in a final volume of 100 μl of RNA binding buffer (10 mM Tris-HCl [pH 7.5], 50 mM NaCl, 1 mM EDTA, and 10 μM ZnCl2 ) supplemented with 1 unit/μl RNasin (Promega), 1 μg/μl heparin (Sigma-Aldrich) and 100 ng/μl yeast tRNA (Roche) for 30 min at 30°C. .. After incubation, 300 μl of Streptavidin MagneSphere paramagnetic particles (Promega) was added for 30 min at 25°C.

    Article Title: ER stress-linked autophagy stabilizes apoptosis effector PERP and triggers its co-localization with SERCA2b at ER–plasma membrane junctions
    Article Snippet: .. The resulting pellet was lysed in Mammalian Lysis Buffer with Protease Inhibitor Cocktail (both from Promega) and 1.5 mg of protein was incubated with the bead–antibody conjugate for 30 min at room temperature. .. Antibody–protein complexes were washed four times in PBS and proteins were removed in SDS sample buffer for Western blot analysis.

    Expressing:

    Article Title: Inhibition of Japanese encephalitis virus infection by the host zinc-finger antiviral protein
    Article Snippet: .. In vitro RNA pull-down assay Different amounts (0, 1, 5, and 10 pmole) of biotin-labeled RNA probe were incubated with 100 μg of ZAP-S-V5 or ZAP-S-del4ZFs expressing A549 cell extracts lysed by CHAPS lysis buffer (10 mM Tris-HCl [pH 7.4], 1 mM MgCl2 , 1 mM EGTA, 0.5% CHAPS, 10% glycerol, and 5 mM 2-mercaptoethanol) containing protease inhibitor cocktail in a final volume of 100 μl of RNA binding buffer (10 mM Tris-HCl [pH 7.5], 50 mM NaCl, 1 mM EDTA, and 10 μM ZnCl2 ) supplemented with 1 unit/μl RNasin (Promega), 1 μg/μl heparin (Sigma-Aldrich) and 100 ng/μl yeast tRNA (Roche) for 30 min at 30°C. .. After incubation, 300 μl of Streptavidin MagneSphere paramagnetic particles (Promega) was added for 30 min at 25°C.

    Western Blot:

    Article Title: miR-512-5p suppresses proliferation, migration and invasion, and induces apoptosis in non-small cell lung cancer cells by targeting ETS1
    Article Snippet: .. Western blot analysis Following transfection, cells of 80% density were lysed using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Haimen, China) containing a Protease Inhibitor Cocktail (Promega Corporation) for 30 min. .. The protein was collected, and the concentrations were measured using a BCA assay kit (Beyotime Institute of Biotechnology).

    Modification:

    Article Title: The Pro-Inflammatory Cytokine, Interleukin-6, Enhances the Polarization of Alternatively Activated Macrophages
    Article Snippet: .. Immunoblotting Briefly, cells were lysed in modified RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS) supplemented with protease inhibitor cocktail (Promega, Madison, WI) and protein concentrations determined via the Bradford assay (Bio-Rad Laboratories Canada, Mississauga, ON, Canada). .. Samples (10–20 µg) were boiled for 10 min with 4x Laemelli buffer, run by SDS-PAGE (4% stacking, 8% separating) and transferred to a nitrocellulose membrane.

    Sonication:

    Article Title: Dopamine D1A directly interacts with otoferlin synaptic pathway proteins: Ca2+ and phosphorylation underlie an NSF-to-AP2mu1 molecular switch
    Article Snippet: .. For co-immunoprecipitation analyses, rat brain tissue was solubilized in calcium-free HBS buffer containing 0.1% Tween-20 and protease inhibitor cocktails (Promega) by brief sonication on ice. .. The supernatant from cold centrifugation at 20 900 × g for 12 min was diluted to a concentration of 1 mg/ml with calcium-free HBS buffer containing a protease inhibitor.

    Lysis:

    Article Title: Identifying Dysregulated Epigenetic Enzyme Activity in Castrate-Resistant Prostate Cancer Development
    Article Snippet: .. Before each assay, cell pellets from PCa cell lines (LAPC4, LNCaP, PC3, DU145, 22RV1, and C4–2) were thawed on ice and resuspended in 300 μ L of lysis buffer; 50 mM Tris-HCl at pH 7.5; 150 mM NaCl; 1% Triton X-100; DNase I (Roche); protease inhibitor cocktail (Promega); and NaF and incubated at RT for 10 min with gentle rotation. ..

    Article Title: Inhibition of Japanese encephalitis virus infection by the host zinc-finger antiviral protein
    Article Snippet: .. In vitro RNA pull-down assay Different amounts (0, 1, 5, and 10 pmole) of biotin-labeled RNA probe were incubated with 100 μg of ZAP-S-V5 or ZAP-S-del4ZFs expressing A549 cell extracts lysed by CHAPS lysis buffer (10 mM Tris-HCl [pH 7.4], 1 mM MgCl2 , 1 mM EGTA, 0.5% CHAPS, 10% glycerol, and 5 mM 2-mercaptoethanol) containing protease inhibitor cocktail in a final volume of 100 μl of RNA binding buffer (10 mM Tris-HCl [pH 7.5], 50 mM NaCl, 1 mM EDTA, and 10 μM ZnCl2 ) supplemented with 1 unit/μl RNasin (Promega), 1 μg/μl heparin (Sigma-Aldrich) and 100 ng/μl yeast tRNA (Roche) for 30 min at 30°C. .. After incubation, 300 μl of Streptavidin MagneSphere paramagnetic particles (Promega) was added for 30 min at 25°C.

    Article Title: ER stress-linked autophagy stabilizes apoptosis effector PERP and triggers its co-localization with SERCA2b at ER–plasma membrane junctions
    Article Snippet: .. The resulting pellet was lysed in Mammalian Lysis Buffer with Protease Inhibitor Cocktail (both from Promega) and 1.5 mg of protein was incubated with the bead–antibody conjugate for 30 min at room temperature. .. Antibody–protein complexes were washed four times in PBS and proteins were removed in SDS sample buffer for Western blot analysis.

    RNA Binding Assay:

    Article Title: Inhibition of Japanese encephalitis virus infection by the host zinc-finger antiviral protein
    Article Snippet: .. In vitro RNA pull-down assay Different amounts (0, 1, 5, and 10 pmole) of biotin-labeled RNA probe were incubated with 100 μg of ZAP-S-V5 or ZAP-S-del4ZFs expressing A549 cell extracts lysed by CHAPS lysis buffer (10 mM Tris-HCl [pH 7.4], 1 mM MgCl2 , 1 mM EGTA, 0.5% CHAPS, 10% glycerol, and 5 mM 2-mercaptoethanol) containing protease inhibitor cocktail in a final volume of 100 μl of RNA binding buffer (10 mM Tris-HCl [pH 7.5], 50 mM NaCl, 1 mM EDTA, and 10 μM ZnCl2 ) supplemented with 1 unit/μl RNasin (Promega), 1 μg/μl heparin (Sigma-Aldrich) and 100 ng/μl yeast tRNA (Roche) for 30 min at 30°C. .. After incubation, 300 μl of Streptavidin MagneSphere paramagnetic particles (Promega) was added for 30 min at 25°C.

    Pull Down Assay:

    Article Title: Inhibition of Japanese encephalitis virus infection by the host zinc-finger antiviral protein
    Article Snippet: .. In vitro RNA pull-down assay Different amounts (0, 1, 5, and 10 pmole) of biotin-labeled RNA probe were incubated with 100 μg of ZAP-S-V5 or ZAP-S-del4ZFs expressing A549 cell extracts lysed by CHAPS lysis buffer (10 mM Tris-HCl [pH 7.4], 1 mM MgCl2 , 1 mM EGTA, 0.5% CHAPS, 10% glycerol, and 5 mM 2-mercaptoethanol) containing protease inhibitor cocktail in a final volume of 100 μl of RNA binding buffer (10 mM Tris-HCl [pH 7.5], 50 mM NaCl, 1 mM EDTA, and 10 μM ZnCl2 ) supplemented with 1 unit/μl RNasin (Promega), 1 μg/μl heparin (Sigma-Aldrich) and 100 ng/μl yeast tRNA (Roche) for 30 min at 30°C. .. After incubation, 300 μl of Streptavidin MagneSphere paramagnetic particles (Promega) was added for 30 min at 25°C.

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  • 90
    Promega trypsin lys c mix mass spec grade
    Identification of glutathionylated proteins in HL-1 cells. HL-1 cells expressing GS M4 were incubated with azido-Ala (0.6 mM) for 24 h and treated with glucose oxidase (GluOx, 6 units) for 10 min. ( A ) In-gel fluorescence detection of glutathionylated proteins. Collected lysates were subjected to click reaction with Cy5-alkyne for fluorescence detection. ( B ) Enrichment and elution of glutathionylated proteins. After click reaction of lysates with biotin-DADPS-alkyne, biotinylated glutathionylated proteins were bound to streptavidin-agarose, washed, eluted in an acidic cleavage condition (10% formic acid), and analyzed by silver staining. ( C ) The number of glutathionylated proteins and peptides under indicated conditions by LC-MS. Biotinylated glutathionylated proteins bound on streptavidin-agarose were digested by <t>trypsin/Lys-C,</t> eluted in an acidic cleavage solution, and analyzed by LC-MS/MS. ( D ) DAVID gene ontology (GO) analysis of identified glutathionylated proteins.
    Trypsin Lys C Mix Mass Spec Grade, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/trypsin lys c mix mass spec grade/product/Promega
    Average 90 stars, based on 14 article reviews
    Price from $9.99 to $1999.99
    trypsin lys c mix mass spec grade - by Bioz Stars, 2020-09
    90/100 stars
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    99
    Promega streptavidin conjugated paramagnetic bead slurry
    RIG-I:RNA conformation as probed by limited trypsin digestion time course. (A to D) RIG-I fragments were detected by Western blotting with a monoclonal antibody to the helicase domain. Cell extracts were incubated with trypsin in the absence (A [–RNA]) or presence of RNA ligands, including nonbinding control yeast tRNA (B [tRNA]), polyI:C (C), or polyU/UC (D), and with 1 mM ATP or AMP-PNP. Aliquots were removed from the reaction at various time points (minutes) post-addition of trypsin. Additional data are provided in Fig. S6 in the supplemental material. (E) Biotinylated RNAs were used in pulldown experiments to test for RNA binding by the 80-kDa and 55-kDa RIG-I fragments. Trypsin digests (+ tryp) and control lysates (− tryp) were incubated without RNA (-RNA), with nonbiotinylated polyU/UC RNA (-btn), with biotinylated polyU/UC RNA (can), or with biotinylated X RNA (X) in the presence of AMP-PNP. After 1.5 h, trypsin digestions were quenched by adding protease inhibitor and incubated with <t>streptavidin</t> paramagnetic beads. RIG-I present in the bead-bound fraction (BND) versus the input fraction (INP) was detected by Western blotting.
    Streptavidin Conjugated Paramagnetic Bead Slurry, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin conjugated paramagnetic bead slurry/product/Promega
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    92
    Promega master mix
    RIG-I:RNA conformation as probed by limited trypsin digestion time course. (A to D) RIG-I fragments were detected by Western blotting with a monoclonal antibody to the helicase domain. Cell extracts were incubated with trypsin in the absence (A [–RNA]) or presence of RNA ligands, including nonbinding control yeast tRNA (B [tRNA]), polyI:C (C), or polyU/UC (D), and with 1 mM ATP or AMP-PNP. Aliquots were removed from the reaction at various time points (minutes) post-addition of trypsin. Additional data are provided in Fig. S6 in the supplemental material. (E) Biotinylated RNAs were used in pulldown experiments to test for RNA binding by the 80-kDa and 55-kDa RIG-I fragments. Trypsin digests (+ tryp) and control lysates (− tryp) were incubated without RNA (-RNA), with nonbiotinylated polyU/UC RNA (-btn), with biotinylated polyU/UC RNA (can), or with biotinylated X RNA (X) in the presence of AMP-PNP. After 1.5 h, trypsin digestions were quenched by adding protease inhibitor and incubated with <t>streptavidin</t> paramagnetic beads. RIG-I present in the bead-bound fraction (BND) versus the input fraction (INP) was detected by Western blotting.
    Master Mix, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/master mix/product/Promega
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    93
    Promega passive lysis buffer
    RIG-I:RNA conformation as probed by limited trypsin digestion time course. (A to D) RIG-I fragments were detected by Western blotting with a monoclonal antibody to the helicase domain. Cell extracts were incubated with trypsin in the absence (A [–RNA]) or presence of RNA ligands, including nonbinding control yeast tRNA (B [tRNA]), polyI:C (C), or polyU/UC (D), and with 1 mM ATP or AMP-PNP. Aliquots were removed from the reaction at various time points (minutes) post-addition of trypsin. Additional data are provided in Fig. S6 in the supplemental material. (E) Biotinylated RNAs were used in pulldown experiments to test for RNA binding by the 80-kDa and 55-kDa RIG-I fragments. Trypsin digests (+ tryp) and control lysates (− tryp) were incubated without RNA (-RNA), with nonbiotinylated polyU/UC RNA (-btn), with biotinylated polyU/UC RNA (can), or with biotinylated X RNA (X) in the presence of AMP-PNP. After 1.5 h, trypsin digestions were quenched by adding protease inhibitor and incubated with <t>streptavidin</t> paramagnetic beads. RIG-I present in the bead-bound fraction (BND) versus the input fraction (INP) was detected by Western blotting.
    Passive Lysis Buffer, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 5054 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/passive lysis buffer/product/Promega
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    Image Search Results


    Identification of glutathionylated proteins in HL-1 cells. HL-1 cells expressing GS M4 were incubated with azido-Ala (0.6 mM) for 24 h and treated with glucose oxidase (GluOx, 6 units) for 10 min. ( A ) In-gel fluorescence detection of glutathionylated proteins. Collected lysates were subjected to click reaction with Cy5-alkyne for fluorescence detection. ( B ) Enrichment and elution of glutathionylated proteins. After click reaction of lysates with biotin-DADPS-alkyne, biotinylated glutathionylated proteins were bound to streptavidin-agarose, washed, eluted in an acidic cleavage condition (10% formic acid), and analyzed by silver staining. ( C ) The number of glutathionylated proteins and peptides under indicated conditions by LC-MS. Biotinylated glutathionylated proteins bound on streptavidin-agarose were digested by trypsin/Lys-C, eluted in an acidic cleavage solution, and analyzed by LC-MS/MS. ( D ) DAVID gene ontology (GO) analysis of identified glutathionylated proteins.

    Journal: Journal of proteome research

    Article Title: Proteomic Identification of Protein Glutathionylation in Cardiomyocytes

    doi: 10.1021/acs.jproteome.8b00986

    Figure Lengend Snippet: Identification of glutathionylated proteins in HL-1 cells. HL-1 cells expressing GS M4 were incubated with azido-Ala (0.6 mM) for 24 h and treated with glucose oxidase (GluOx, 6 units) for 10 min. ( A ) In-gel fluorescence detection of glutathionylated proteins. Collected lysates were subjected to click reaction with Cy5-alkyne for fluorescence detection. ( B ) Enrichment and elution of glutathionylated proteins. After click reaction of lysates with biotin-DADPS-alkyne, biotinylated glutathionylated proteins were bound to streptavidin-agarose, washed, eluted in an acidic cleavage condition (10% formic acid), and analyzed by silver staining. ( C ) The number of glutathionylated proteins and peptides under indicated conditions by LC-MS. Biotinylated glutathionylated proteins bound on streptavidin-agarose were digested by trypsin/Lys-C, eluted in an acidic cleavage solution, and analyzed by LC-MS/MS. ( D ) DAVID gene ontology (GO) analysis of identified glutathionylated proteins.

    Article Snippet: Other materials were purchased from the following vendors: biotin-DADPS-alkyne, biotin-DDE-alkyne, biotin-PC-alkyne and THPTA (Click Chemistry Tools), glucose oxidase (G2133), HL-1 cell line (SCC065), Claycomb medium (51800C), norepinephrine (A0937) (Sigma), Polybrene (Millipore), fetal bovine serum (HyClone), penicillin and streptomycin (Gibco), streptavidin-agarose (20359, Pierce), protease-inhibitor cocktail (A32955, Pierce), and Trypsin/Lys-C Mass spec grade (V5072, Promega).

    Techniques: Expressing, Incubation, Fluorescence, Silver Staining, Liquid Chromatography with Mass Spectroscopy

    RIG-I:RNA conformation as probed by limited trypsin digestion time course. (A to D) RIG-I fragments were detected by Western blotting with a monoclonal antibody to the helicase domain. Cell extracts were incubated with trypsin in the absence (A [–RNA]) or presence of RNA ligands, including nonbinding control yeast tRNA (B [tRNA]), polyI:C (C), or polyU/UC (D), and with 1 mM ATP or AMP-PNP. Aliquots were removed from the reaction at various time points (minutes) post-addition of trypsin. Additional data are provided in Fig. S6 in the supplemental material. (E) Biotinylated RNAs were used in pulldown experiments to test for RNA binding by the 80-kDa and 55-kDa RIG-I fragments. Trypsin digests (+ tryp) and control lysates (− tryp) were incubated without RNA (-RNA), with nonbiotinylated polyU/UC RNA (-btn), with biotinylated polyU/UC RNA (can), or with biotinylated X RNA (X) in the presence of AMP-PNP. After 1.5 h, trypsin digestions were quenched by adding protease inhibitor and incubated with streptavidin paramagnetic beads. RIG-I present in the bead-bound fraction (BND) versus the input fraction (INP) was detected by Western blotting.

    Journal: mBio

    Article Title: RNAs Containing Modified Nucleotides Fail To Trigger RIG-I Conformational Changes for Innate Immune Signaling

    doi: 10.1128/mBio.00833-16

    Figure Lengend Snippet: RIG-I:RNA conformation as probed by limited trypsin digestion time course. (A to D) RIG-I fragments were detected by Western blotting with a monoclonal antibody to the helicase domain. Cell extracts were incubated with trypsin in the absence (A [–RNA]) or presence of RNA ligands, including nonbinding control yeast tRNA (B [tRNA]), polyI:C (C), or polyU/UC (D), and with 1 mM ATP or AMP-PNP. Aliquots were removed from the reaction at various time points (minutes) post-addition of trypsin. Additional data are provided in Fig. S6 in the supplemental material. (E) Biotinylated RNAs were used in pulldown experiments to test for RNA binding by the 80-kDa and 55-kDa RIG-I fragments. Trypsin digests (+ tryp) and control lysates (− tryp) were incubated without RNA (-RNA), with nonbiotinylated polyU/UC RNA (-btn), with biotinylated polyU/UC RNA (can), or with biotinylated X RNA (X) in the presence of AMP-PNP. After 1.5 h, trypsin digestions were quenched by adding protease inhibitor and incubated with streptavidin paramagnetic beads. RIG-I present in the bead-bound fraction (BND) versus the input fraction (INP) was detected by Western blotting.

    Article Snippet: After 30 min, reaction mixtures were supplemented with 150 µl of streptavidin-conjugated paramagnetic bead slurry (Promega) and incubated at room temperature for 1 h with rocking.

    Techniques: Western Blot, Incubation, RNA Binding Assay, Protease Inhibitor

    RNA mod and RIG-I binding affinity. (A) Radiolabeled polyU/UC RNA was incubated with purified recombinant RIG-I to allow complex formation and then applied to a nitrocellulose membrane filter, which retains RNA-protein complexes, while unbound RNA passes through the membrane. The fraction of bound RNA was normalized to the maximum observed signal. Combined data from at least three independent experiments per ligand are presented with solid lines indicating the best-fit nonlinear regression and dashed lines indicating 95% confidence intervals. (B) Calculated equilibrium binding dissociation constants ( K d ) derived from data shown in panel A, including 95% confidence interval, goodness of fit ( R 2 ), and statistical test ( P value), demonstrating a significant difference between the two curve parameters K d and slope for each comparison of RNA mod versus RNA can . For the m6A data (indicated by an asterisk [*]), the experimental maximum observed signal was normalized to 100% RNA bound, although a binding plateau was not observed. Therefore, the accurate RIG-I:RNA m6A binding constant is likely higher (lower affinity) than that presented. Additional data are provided in Fig. S5 in the supplemental material. (C) Biotinylated polyU/UC RNA with the indicated modified nucleotides was added to Huh7 cell lysate. Negative controls included biotinylated X-RNA (X) and nonbiotinylated polyU/UC (-btn). RIG-I:RNA complexes that were captured with streptavidin-conjugated paramagnetic beads (BND) were detected by Western blotting with anti-RIG-I, relative to the RIG-I in 10% input (INP). The signal was quantified in the BND fraction relative to the INP fraction and normalized to canonical RNA (100%).

    Journal: mBio

    Article Title: RNAs Containing Modified Nucleotides Fail To Trigger RIG-I Conformational Changes for Innate Immune Signaling

    doi: 10.1128/mBio.00833-16

    Figure Lengend Snippet: RNA mod and RIG-I binding affinity. (A) Radiolabeled polyU/UC RNA was incubated with purified recombinant RIG-I to allow complex formation and then applied to a nitrocellulose membrane filter, which retains RNA-protein complexes, while unbound RNA passes through the membrane. The fraction of bound RNA was normalized to the maximum observed signal. Combined data from at least three independent experiments per ligand are presented with solid lines indicating the best-fit nonlinear regression and dashed lines indicating 95% confidence intervals. (B) Calculated equilibrium binding dissociation constants ( K d ) derived from data shown in panel A, including 95% confidence interval, goodness of fit ( R 2 ), and statistical test ( P value), demonstrating a significant difference between the two curve parameters K d and slope for each comparison of RNA mod versus RNA can . For the m6A data (indicated by an asterisk [*]), the experimental maximum observed signal was normalized to 100% RNA bound, although a binding plateau was not observed. Therefore, the accurate RIG-I:RNA m6A binding constant is likely higher (lower affinity) than that presented. Additional data are provided in Fig. S5 in the supplemental material. (C) Biotinylated polyU/UC RNA with the indicated modified nucleotides was added to Huh7 cell lysate. Negative controls included biotinylated X-RNA (X) and nonbiotinylated polyU/UC (-btn). RIG-I:RNA complexes that were captured with streptavidin-conjugated paramagnetic beads (BND) were detected by Western blotting with anti-RIG-I, relative to the RIG-I in 10% input (INP). The signal was quantified in the BND fraction relative to the INP fraction and normalized to canonical RNA (100%).

    Article Snippet: After 30 min, reaction mixtures were supplemented with 150 µl of streptavidin-conjugated paramagnetic bead slurry (Promega) and incubated at room temperature for 1 h with rocking.

    Techniques: Binding Assay, Incubation, Purification, Recombinant, Derivative Assay, Modification, Western Blot