Protein Extraction:Article Title: Sex-specific effects of acetylation on tauopathy in aging htau mice.
Article Snippet: .. Proteins from the pre-frontal cortex region of the brain homogenate were extracted using the total protein extraction reagent (TPER buffer, 25 mM bicine 150 mM sodium chloride, pH 7.6, ThermoFisher Scientific, Waltham MA; Cat# 78510) supplemented with protease (A32963), phosphatase (A32957), and deacetylase inhibitors (5 μM trichostatin). .. Equal protein amounts (15–30 μg) were separated on 4–20% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels (Bio-Rad, Hercules, CA) at 120 V for 120 min and then electro-transferred to polyvinylidene difluoride membranes at 100 V for 60 min.
Article Title: Sex-specific effects of acetylation on tauopathy in aging htau mice
Article Snippet: .. Proteins from the pre-frontal cortex region of the brain homogenate were extracted using the total protein extraction reagent (TPER buffer, 25 mM bicine 150 mM sodium chloride, pH 7.6, ThermoFisher Scientific, Waltham MA; Cat# 78510) supplemented with protease (A32963), phosphatase (A32957), and deacetylase inhibitors (5 μM trichostatin). .. Equal protein amounts (15–30 μg) were separated on 4–20% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels (Bio-Rad, Hercules, CA) at 120 V for 120 min and then electro-transferred to polyvinylidene difluoride membranes at 100 V for 60 min.
Histone Deacetylase Assay:Article Title: Sex-specific effects of acetylation on tauopathy in aging htau mice.
Article Snippet: .. Proteins from the pre-frontal cortex region of the brain homogenate were extracted using the total protein extraction reagent (TPER buffer, 25 mM bicine 150 mM sodium chloride, pH 7.6, ThermoFisher Scientific, Waltham MA; Cat# 78510) supplemented with protease (A32963), phosphatase (A32957), and deacetylase inhibitors (5 μM trichostatin). .. Equal protein amounts (15–30 μg) were separated on 4–20% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels (Bio-Rad, Hercules, CA) at 120 V for 120 min and then electro-transferred to polyvinylidene difluoride membranes at 100 V for 60 min.
Article Title: Sex-specific effects of acetylation on tauopathy in aging htau mice
Article Snippet: .. Proteins from the pre-frontal cortex region of the brain homogenate were extracted using the total protein extraction reagent (TPER buffer, 25 mM bicine 150 mM sodium chloride, pH 7.6, ThermoFisher Scientific, Waltham MA; Cat# 78510) supplemented with protease (A32963), phosphatase (A32957), and deacetylase inhibitors (5 μM trichostatin). .. Equal protein amounts (15–30 μg) were separated on 4–20% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels (Bio-Rad, Hercules, CA) at 120 V for 120 min and then electro-transferred to polyvinylidene difluoride membranes at 100 V for 60 min.
Western Blot:Article Title: Reprogramming encapsulins into modular carbon-fixing nanocompartments
Article Snippet: .. For immunoblotting, PAGE separated proteins were transferred onto nitrocellulose Hybond-N membrane (Cytiva) in transfer buffer (25 mM bicine, 25 mM Bis-Tris, 1 mM EDTA) using a XCell IITM Blot Module (Invitrogen) at 25 V, 4 °C for 1 h. The membranes were then incubated in blocking reagent (5% w/v skim milk in TBS; 50 mM Tris-HCl pH 8.0, 150 mM NaCl) for 1 hour at room temperature with gentle orbital rotation. ..
Polyacrylamide Gel Electrophoresis:Article Title: Reprogramming encapsulins into modular carbon-fixing nanocompartments
Article Snippet: .. For immunoblotting, PAGE separated proteins were transferred onto nitrocellulose Hybond-N membrane (Cytiva) in transfer buffer (25 mM bicine, 25 mM Bis-Tris, 1 mM EDTA) using a XCell IITM Blot Module (Invitrogen) at 25 V, 4 °C for 1 h. The membranes were then incubated in blocking reagent (5% w/v skim milk in TBS; 50 mM Tris-HCl pH 8.0, 150 mM NaCl) for 1 hour at room temperature with gentle orbital rotation. ..
Membrane:Article Title: Reprogramming encapsulins into modular carbon-fixing nanocompartments
Article Snippet: .. For immunoblotting, PAGE separated proteins were transferred onto nitrocellulose Hybond-N membrane (Cytiva) in transfer buffer (25 mM bicine, 25 mM Bis-Tris, 1 mM EDTA) using a XCell IITM Blot Module (Invitrogen) at 25 V, 4 °C for 1 h. The membranes were then incubated in blocking reagent (5% w/v skim milk in TBS; 50 mM Tris-HCl pH 8.0, 150 mM NaCl) for 1 hour at room temperature with gentle orbital rotation. ..
Incubation:Article Title: Reprogramming encapsulins into modular carbon-fixing nanocompartments
Article Snippet: .. For immunoblotting, PAGE separated proteins were transferred onto nitrocellulose Hybond-N membrane (Cytiva) in transfer buffer (25 mM bicine, 25 mM Bis-Tris, 1 mM EDTA) using a XCell IITM Blot Module (Invitrogen) at 25 V, 4 °C for 1 h. The membranes were then incubated in blocking reagent (5% w/v skim milk in TBS; 50 mM Tris-HCl pH 8.0, 150 mM NaCl) for 1 hour at room temperature with gentle orbital rotation. ..
Article Title: Pan-modification profiling facilitates a cross-evolutionary dissection of the thermoregulated ribosomal epitranscriptome.
Article Snippet: .. The samples were then purified by ethanol precipitation. (6) CMC treatment was performed in 30 μL of 0.17 M CMC in BEU buffer (50 mM bicine, pH 8.3, 4 mM EDTA, and 7 M urea), essentially as in,19 except cleanups following the CMC treatment and sodium bicarbonate incubation were performed on Dyna- beads MyOne Silane beads (Thermo Fisher scientific). (7) AlkB WT and D1355 mutant mix was carried out essentially as in.52 RNA was incubated in a 50 μL reaction with 40 pmol AlkB WT and 80 pmol AlkB D135S mutant protein in buffer containing 300 mM KCl, 2 mM MgCl2, 50 μM of (NH4)2Fe(SO4)2⋅6H2O, 300 μM 2-ketoglutarate (2-KG), 2 mM l-ascorbic acid, 50 μg/ml BSA, and 50 mM MES buffer (pH 5.0) for 2 hours at room temperature, and quenched using 5mM EDTA. .. The demethylated RNAs were purified by the Zymo Research RNA Clean & Concentrator-5 kit (Zymo research). (8) NaHCO3 treatment was carried out essentially as in Li et al. 63 2/12 of the RNA pool (a double portion relative to other treat- ments, to account for enhanced degradation due to the conditions of the treatment) were incubated in alkaline buffer (0.1 M NaHCO3, 5 mM EDTA, pH 10.2) for 1 hour in 65 ◦C.
Blocking Assay:Article Title: Reprogramming encapsulins into modular carbon-fixing nanocompartments
Article Snippet: .. For immunoblotting, PAGE separated proteins were transferred onto nitrocellulose Hybond-N membrane (Cytiva) in transfer buffer (25 mM bicine, 25 mM Bis-Tris, 1 mM EDTA) using a XCell IITM Blot Module (Invitrogen) at 25 V, 4 °C for 1 h. The membranes were then incubated in blocking reagent (5% w/v skim milk in TBS; 50 mM Tris-HCl pH 8.0, 150 mM NaCl) for 1 hour at room temperature with gentle orbital rotation. ..
Gentle:Article Title: Reprogramming encapsulins into modular carbon-fixing nanocompartments
Article Snippet: .. For immunoblotting, PAGE separated proteins were transferred onto nitrocellulose Hybond-N membrane (Cytiva) in transfer buffer (25 mM bicine, 25 mM Bis-Tris, 1 mM EDTA) using a XCell IITM Blot Module (Invitrogen) at 25 V, 4 °C for 1 h. The membranes were then incubated in blocking reagent (5% w/v skim milk in TBS; 50 mM Tris-HCl pH 8.0, 150 mM NaCl) for 1 hour at room temperature with gentle orbital rotation. ..
Protease Inhibitor:Article Title: Rapid Biochemical Analysis of Postmortem Serum and Myocardial Homogenates—An Exploratory Study
Article Snippet: .. In the main study, the myocardial homogenates were prepared at a 1:20 ratio (200 mg tissue: 4 mL buffer) in dH 2 O, T-PER (a proprietary detergent in 25 mM bicine, 150 mM NaCl; pH 7.6, Thermo Fisher Scientific, Vantaa, Finland, #78510), and Tris-Urea (25 mM Tris-HCl), or 2 M Urea (Thermo Fisher Scientific, #424585000; pH 7.4) buffers supplemented with HALT protease inhibitor cocktail (Thermo Fisher Scientific, #78438). ..
Purification:Article Title: Pan-modification profiling facilitates a cross-evolutionary dissection of the thermoregulated ribosomal epitranscriptome.
Article Snippet: .. The samples were then purified by ethanol precipitation. (6) CMC treatment was performed in 30 μL of 0.17 M CMC in BEU buffer (50 mM bicine, pH 8.3, 4 mM EDTA, and 7 M urea), essentially as in,19 except cleanups following the CMC treatment and sodium bicarbonate incubation were performed on Dyna- beads MyOne Silane beads (Thermo Fisher scientific). (7) AlkB WT and D1355 mutant mix was carried out essentially as in.52 RNA was incubated in a 50 μL reaction with 40 pmol AlkB WT and 80 pmol AlkB D135S mutant protein in buffer containing 300 mM KCl, 2 mM MgCl2, 50 μM of (NH4)2Fe(SO4)2⋅6H2O, 300 μM 2-ketoglutarate (2-KG), 2 mM l-ascorbic acid, 50 μg/ml BSA, and 50 mM MES buffer (pH 5.0) for 2 hours at room temperature, and quenched using 5mM EDTA. .. The demethylated RNAs were purified by the Zymo Research RNA Clean & Concentrator-5 kit (Zymo research). (8) NaHCO3 treatment was carried out essentially as in Li et al. 63 2/12 of the RNA pool (a double portion relative to other treat- ments, to account for enhanced degradation due to the conditions of the treatment) were incubated in alkaline buffer (0.1 M NaHCO3, 5 mM EDTA, pH 10.2) for 1 hour in 65 ◦C.
Article Title: Solution-state nuclear magnetic resonance studies of the Salmonella typhimurium tryptophan synthase complex
Article Snippet: .. Purified proteins, including St TS, StαTS and StβTS were buffer exchanged into D 2 O-based NMR buffer (0.05 mM bicine, pH 7.80, 1 mM EDTA, 0.02 mM PLP, and 10 mM BME in D 2 O) using ZEBA desalting columns (Thermo Fischer). .. The StTS complex concentration was 850 μM, and StαTS and StβTS ranged from 150 to 300 μM, with 10 mM indole (Thermo Fischer), 10 mM G3P, 10 mM tryptophan, or 66 mM serine, where appropriate.
Ethanol Precipitation:Article Title: Pan-modification profiling facilitates a cross-evolutionary dissection of the thermoregulated ribosomal epitranscriptome.
Article Snippet: .. The samples were then purified by ethanol precipitation. (6) CMC treatment was performed in 30 μL of 0.17 M CMC in BEU buffer (50 mM bicine, pH 8.3, 4 mM EDTA, and 7 M urea), essentially as in,19 except cleanups following the CMC treatment and sodium bicarbonate incubation were performed on Dyna- beads MyOne Silane beads (Thermo Fisher scientific). (7) AlkB WT and D1355 mutant mix was carried out essentially as in.52 RNA was incubated in a 50 μL reaction with 40 pmol AlkB WT and 80 pmol AlkB D135S mutant protein in buffer containing 300 mM KCl, 2 mM MgCl2, 50 μM of (NH4)2Fe(SO4)2⋅6H2O, 300 μM 2-ketoglutarate (2-KG), 2 mM l-ascorbic acid, 50 μg/ml BSA, and 50 mM MES buffer (pH 5.0) for 2 hours at room temperature, and quenched using 5mM EDTA. .. The demethylated RNAs were purified by the Zymo Research RNA Clean & Concentrator-5 kit (Zymo research). (8) NaHCO3 treatment was carried out essentially as in Li et al. 63 2/12 of the RNA pool (a double portion relative to other treat- ments, to account for enhanced degradation due to the conditions of the treatment) were incubated in alkaline buffer (0.1 M NaHCO3, 5 mM EDTA, pH 10.2) for 1 hour in 65 ◦C.
CMC:Article Title: Pan-modification profiling facilitates a cross-evolutionary dissection of the thermoregulated ribosomal epitranscriptome.
Article Snippet: .. The samples were then purified by ethanol precipitation. (6) CMC treatment was performed in 30 μL of 0.17 M CMC in BEU buffer (50 mM bicine, pH 8.3, 4 mM EDTA, and 7 M urea), essentially as in,19 except cleanups following the CMC treatment and sodium bicarbonate incubation were performed on Dyna- beads MyOne Silane beads (Thermo Fisher scientific). (7) AlkB WT and D1355 mutant mix was carried out essentially as in.52 RNA was incubated in a 50 μL reaction with 40 pmol AlkB WT and 80 pmol AlkB D135S mutant protein in buffer containing 300 mM KCl, 2 mM MgCl2, 50 μM of (NH4)2Fe(SO4)2⋅6H2O, 300 μM 2-ketoglutarate (2-KG), 2 mM l-ascorbic acid, 50 μg/ml BSA, and 50 mM MES buffer (pH 5.0) for 2 hours at room temperature, and quenched using 5mM EDTA. .. The demethylated RNAs were purified by the Zymo Research RNA Clean & Concentrator-5 kit (Zymo research). (8) NaHCO3 treatment was carried out essentially as in Li et al. 63 2/12 of the RNA pool (a double portion relative to other treat- ments, to account for enhanced degradation due to the conditions of the treatment) were incubated in alkaline buffer (0.1 M NaHCO3, 5 mM EDTA, pH 10.2) for 1 hour in 65 ◦C.
Mutagenesis:Article Title: Pan-modification profiling facilitates a cross-evolutionary dissection of the thermoregulated ribosomal epitranscriptome.
Article Snippet: .. The samples were then purified by ethanol precipitation. (6) CMC treatment was performed in 30 μL of 0.17 M CMC in BEU buffer (50 mM bicine, pH 8.3, 4 mM EDTA, and 7 M urea), essentially as in,19 except cleanups following the CMC treatment and sodium bicarbonate incubation were performed on Dyna- beads MyOne Silane beads (Thermo Fisher scientific). (7) AlkB WT and D1355 mutant mix was carried out essentially as in.52 RNA was incubated in a 50 μL reaction with 40 pmol AlkB WT and 80 pmol AlkB D135S mutant protein in buffer containing 300 mM KCl, 2 mM MgCl2, 50 μM of (NH4)2Fe(SO4)2⋅6H2O, 300 μM 2-ketoglutarate (2-KG), 2 mM l-ascorbic acid, 50 μg/ml BSA, and 50 mM MES buffer (pH 5.0) for 2 hours at room temperature, and quenched using 5mM EDTA. .. The demethylated RNAs were purified by the Zymo Research RNA Clean & Concentrator-5 kit (Zymo research). (8) NaHCO3 treatment was carried out essentially as in Li et al. 63 2/12 of the RNA pool (a double portion relative to other treat- ments, to account for enhanced degradation due to the conditions of the treatment) were incubated in alkaline buffer (0.1 M NaHCO3, 5 mM EDTA, pH 10.2) for 1 hour in 65 ◦C.
Nuclear Magnetic Resonance:Article Title: Solution-state nuclear magnetic resonance studies of the Salmonella typhimurium tryptophan synthase complex
Article Snippet: .. Purified proteins, including St TS, StαTS and StβTS were buffer exchanged into D 2 O-based NMR buffer (0.05 mM bicine, pH 7.80, 1 mM EDTA, 0.02 mM PLP, and 10 mM BME in D 2 O) using ZEBA desalting columns (Thermo Fischer). .. The StTS complex concentration was 850 μM, and StαTS and StβTS ranged from 150 to 300 μM, with 10 mM indole (Thermo Fischer), 10 mM G3P, 10 mM tryptophan, or 66 mM serine, where appropriate.
Plasmid Purification:Article Title: Solution-state nuclear magnetic resonance studies of the Salmonella typhimurium tryptophan synthase complex
Article Snippet: .. Purified proteins, including St TS, StαTS and StβTS were buffer exchanged into D 2 O-based NMR buffer (0.05 mM bicine, pH 7.80, 1 mM EDTA, 0.02 mM PLP, and 10 mM BME in D 2 O) using ZEBA desalting columns (Thermo Fischer). .. The StTS complex concentration was 850 μM, and StαTS and StβTS ranged from 150 to 300 μM, with 10 mM indole (Thermo Fischer), 10 mM G3P, 10 mM tryptophan, or 66 mM serine, where appropriate.
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