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Differential binding of wild-type and mutant CovR. Biotinylated promoter regions of gbpC and SMU.1882 were immobilized on streptavidin biosensors and then exposed to 0.5 µM CovR, CovR D53A and CovR D53E proteins purified from E. coli as described in the text. The reactions were carried out in binding buffer [20 mM <t>Tris,</t> 100 mM NaCl, 0.01 mM DTT, 5 % glycerol (v/v), 1 mM <t>EDTA,</t> 0.01 mg ml −1 BSA, 5 mM MgCl 2 and 10 µg ml −1 poly (dI-dC) at pH 7.5] for a period of 5 min to allow association followed by 2 min exposure to binding buffer to allow dissociation.
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1) Product Images from "Role of CovR phosphorylation in gene transcription in Streptococcus mutans"

Article Title: Role of CovR phosphorylation in gene transcription in Streptococcus mutans

Journal: Microbiology

doi: 10.1099/mic.0.000641

Differential binding of wild-type and mutant CovR. Biotinylated promoter regions of gbpC and SMU.1882 were immobilized on streptavidin biosensors and then exposed to 0.5 µM CovR, CovR D53A and CovR D53E proteins purified from E. coli as described in the text. The reactions were carried out in binding buffer [20 mM Tris, 100 mM NaCl, 0.01 mM DTT, 5 % glycerol (v/v), 1 mM EDTA, 0.01 mg ml −1 BSA, 5 mM MgCl 2 and 10 µg ml −1 poly (dI-dC) at pH 7.5] for a period of 5 min to allow association followed by 2 min exposure to binding buffer to allow dissociation.
Figure Legend Snippet: Differential binding of wild-type and mutant CovR. Biotinylated promoter regions of gbpC and SMU.1882 were immobilized on streptavidin biosensors and then exposed to 0.5 µM CovR, CovR D53A and CovR D53E proteins purified from E. coli as described in the text. The reactions were carried out in binding buffer [20 mM Tris, 100 mM NaCl, 0.01 mM DTT, 5 % glycerol (v/v), 1 mM EDTA, 0.01 mg ml −1 BSA, 5 mM MgCl 2 and 10 µg ml −1 poly (dI-dC) at pH 7.5] for a period of 5 min to allow association followed by 2 min exposure to binding buffer to allow dissociation.

Techniques Used: Binding Assay, Mutagenesis, Purification

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Clone Assay:

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Centrifugation:

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Filtration:

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Blocking Assay:

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Electrophoresis:

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Incubation:

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Infection:

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Western Blot:

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Flow Cytometry:

Article Title: Structures of chaperone-substrate complexes docked onto the export gate in a type III secretion system
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Protease Inhibitor:

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Article Title: Amino acids disrupt calcium-dependent adhesion of stratum corneum
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Article Title: ATP Synthase C-Subunit-Deficient Mitochondria Have a Small Cyclosporine A-Sensitive Channel, but Lack the Permeability Transition Pore
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Article Title: ISWI chromatin remodellers sense nucleosome modifications to determine substrate preference
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Article Title: Expression and purification of the functional ectodomain of human anthrax toxin receptor 2 in E. coli Origami B cells with assistance of bacterial Trigger Factor
Article Snippet: .. Bacterial cells were harvested by centrifugation and the cell paste was re-suspended in Buffer A (20 mM TrisHCl pH 7.3, 300 mM NaCl, 10mM imidazole) with 1mg/ml of lysozyme and EDTA-free protease inhibitor cocktail (1 tablet/100 ml) (Sigma). ..

Article Title: USP48 restrains resection by site-specific cleavage of the BRCA1 ubiquitin mark from H2A
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Article Title: Protective effect of stromal Dickkopf-3 in prostate cancer: opposing roles for TGFBI and ECM-1
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Article Title: SIRT7 is an RNA-activated protein lysine deacylase
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Article Title: Targeting lonidamine to mitochondria mitigates lung tumorigenesis and brain metastasis
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Article Title: New Use for CETSA: Monitoring Innate Immune Receptor Stability via Post-Translational Modification by OGT
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Article Title: Structures of chaperone-substrate complexes docked onto the export gate in a type III secretion system
Article Snippet: .. Unlabeled proteins were expressed in BL21(DE3) cells grown in Luria-Bertani (LB) medium in the presence of ampicillin (100 μg ml−1 ) at 37 ℃, and protein expression was induced at 18 ℃ with 0.4 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) at OD600 ≈ 0.5 for ~48 h. Cells were harvested at OD600 ≈ 1.5 and were suspended in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1% EDTA-free protease inhibitor cocktail (Sigma-Aldrich), and 20 mM imidazole. .. Cells were disrupted by a high-pressure homogenizer and centrifuged at 20,000 r.p.m. for 1 h. Proteins were purified using Ni Sepharose 6 Fast Flow resin (GE Healthcare), followed by tag removal by TEV protease at 4 ℃ for 12–20 h and gel filtration using Superdex 75 16/60.

Article Title: Visualizing conformational dynamics of proteins in solution and at the cell membrane
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Cell Culture:

Article Title: Expression and purification of the functional ectodomain of human anthrax toxin receptor 2 in E. coli Origami B cells with assistance of bacterial Trigger Factor
Article Snippet: The Origami B (DE3) cells harboring pCOLD-TF-R318 were cultured in a 200 ml of LB medium at 30 ºC for overnight. .. Bacterial cells were harvested by centrifugation and the cell paste was re-suspended in Buffer A (20 mM TrisHCl pH 7.3, 300 mM NaCl, 10mM imidazole) with 1mg/ml of lysozyme and EDTA-free protease inhibitor cocktail (1 tablet/100 ml) (Sigma).

Article Title: SIRT7 is an RNA-activated protein lysine deacylase
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Protein Concentration:

Article Title: Structures of chaperone-substrate complexes docked onto the export gate in a type III secretion system
Article Snippet: Unlabeled proteins were expressed in BL21(DE3) cells grown in Luria-Bertani (LB) medium in the presence of ampicillin (100 μg ml−1 ) at 37 ℃, and protein expression was induced at 18 ℃ with 0.4 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) at OD600 ≈ 0.5 for ~48 h. Cells were harvested at OD600 ≈ 1.5 and were suspended in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1% EDTA-free protease inhibitor cocktail (Sigma-Aldrich), and 20 mM imidazole. .. Protein concentration was determined spectrophotometrically at 280 nm using the corresponding extinction coefficient.

Sonication:

Article Title: The SMX DNA Repair Tri-nuclease
Article Snippet: .. Cells were resuspended in HisTRAP buffer (25 mM sodium phosphate pH 7.8, 500 mM NaCl, 10% glycerol, 0.05% NP-40, 1 mM DTT, 5 mM imidazole) supplemented with EDTA-Free Protease Inhibitor Cocktail (Sigma) and lysed by sonication on ice (5 × 15 s with 45 s incubations on ice in between) using a Soniprep 150 (MSE) at 50% maximum setting. .. Insoluble material was removed by ultracentrifugation in a Beckman Coulter Optima LE-80K Ultracentrifuge with the Type 45 Ti rotor for 1 hr 15 min at 35,000 rpm (4°C).

Article Title: USP48 restrains resection by site-specific cleavage of the BRCA1 ubiquitin mark from H2A
Article Snippet: Cells were infected at a density of 1 × 106 cells per ml and grown for 72 h. Cells were collected in lysis buffer (50 mM TRIS pH 8, 150 mM NaCl, 2 mM TCEP) and complete EDTA-free protease inhibitor (Sigma) was added. .. Cells were lysed by sonication and sample was cleared by centrifugation at 21,000 × g for 30 min. Supernatant was loaded on His-affinity column (GE Healthcare) and column was washed with lysis buffer + 50 mM Imidazol.

Article Title: USP48 restrains resection by site-specific cleavage of the BRCA1 ubiquitin mark from H2A
Article Snippet: .. Cells were collected in lysis buffer (50 mM TRIS pH 8, 100 mM NaCl, 1 mM β-mercaptoethanol) with Complete EDTA-free protease inhibitor (Sigma) and lysed by sonication. .. Lysate was cleared by spinning down at 21,000 × g and loaded on TALON beads (Clontech Laboratories, Inc.).

Article Title: Targeting lonidamine to mitochondria mitigates lung tumorigenesis and brain metastasis
Article Snippet: .. Autophagy assessment in vivo by Western blot analysis Lung tissues harvested at necropsy were processed into protein lysates by homogenization (Bio-Gen PRO200, PRO Scientific Inc., Oxford, CT) and sonication (50 watt sonic dismembrator, Fisher Scientific, Hampton, NH) in T-PER® Tissue Protein Extraction Reagent (Thermo Fisher Scientific) with cOmplete™ EDTA-free protease inhibitor cocktail and PhosSTOP phosphatase inhibitors (Sigma-Aldrich) according to manufacturer’s instructions. .. Following centrifugation to remove insoluble material, protein levels in soluble lysates were quantified using the DC protein assay (Bio-Rad) and 15 µg/lane was loaded onto precast 4–20% Mini-Protean and Criterion TGX gels (Bio-Rad).

Article Title: Visualizing conformational dynamics of proteins in solution and at the cell membrane
Article Snippet: For fluorometry experiments, between 1 and 4 frozen pellets were thawed and resuspended in 0.5–0.8 mL Stabilization Buffer Tris (SBT) (in mM: KCl 70; MgCl2 1; Trizma Base 30; pH 7.4) supplemented with cOmplete mini EDTA-free protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). .. The suspension was sonicated using a Sonifier 450 with MicroTip (Branson, Danbury, CT) with settings of power = 4 and duty cycle = 50% for a total of 10 pulses.

Injection:

Article Title: USP48 restrains resection by site-specific cleavage of the BRCA1 ubiquitin mark from H2A
Article Snippet: Cells were collected in lysis buffer (50 mM TRIS pH 8, 100 mM NaCl, 1 mM β-mercaptoethanol) with Complete EDTA-free protease inhibitor (Sigma) and lysed by sonication. .. Sample was diluted to 50 mM NaCl and injected into a Resource Q anion exchange column (GE Healthcare).

Recombinant:

Article Title: ISWI chromatin remodellers sense nucleosome modifications to determine substrate preference
Article Snippet: Paragraph title: Production and purification of recombinant histones ... Cell pellets were resuspended in 10 ml of cold lysis buffer (50 mM Tris, 100 mM NaCl, 1 mM EDTA, 1 mM 2-mercaptoethanol, pH 7.6 at 4 °C) with cOmplete, EDTA-free Protease Inhibitor Cocktail (Sigma-Aldrich) per litre of culture and homogenized by passage several times through an 18-gauge needle.

DC Protein Assay:

Article Title: Targeting lonidamine to mitochondria mitigates lung tumorigenesis and brain metastasis
Article Snippet: Autophagy assessment in vivo by Western blot analysis Lung tissues harvested at necropsy were processed into protein lysates by homogenization (Bio-Gen PRO200, PRO Scientific Inc., Oxford, CT) and sonication (50 watt sonic dismembrator, Fisher Scientific, Hampton, NH) in T-PER® Tissue Protein Extraction Reagent (Thermo Fisher Scientific) with cOmplete™ EDTA-free protease inhibitor cocktail and PhosSTOP phosphatase inhibitors (Sigma-Aldrich) according to manufacturer’s instructions. .. Following centrifugation to remove insoluble material, protein levels in soluble lysates were quantified using the DC protein assay (Bio-Rad) and 15 µg/lane was loaded onto precast 4–20% Mini-Protean and Criterion TGX gels (Bio-Rad).

In Vivo:

Article Title: Targeting lonidamine to mitochondria mitigates lung tumorigenesis and brain metastasis
Article Snippet: .. Autophagy assessment in vivo by Western blot analysis Lung tissues harvested at necropsy were processed into protein lysates by homogenization (Bio-Gen PRO200, PRO Scientific Inc., Oxford, CT) and sonication (50 watt sonic dismembrator, Fisher Scientific, Hampton, NH) in T-PER® Tissue Protein Extraction Reagent (Thermo Fisher Scientific) with cOmplete™ EDTA-free protease inhibitor cocktail and PhosSTOP phosphatase inhibitors (Sigma-Aldrich) according to manufacturer’s instructions. .. Following centrifugation to remove insoluble material, protein levels in soluble lysates were quantified using the DC protein assay (Bio-Rad) and 15 µg/lane was loaded onto precast 4–20% Mini-Protean and Criterion TGX gels (Bio-Rad).

Mutagenesis:

Article Title: Structures of chaperone-substrate complexes docked onto the export gate in a type III secretion system
Article Snippet: All mutants were constructed by site-directed mutagenesis using PfuTurbo high-fidelity DNA polymerase (Agilent). .. Unlabeled proteins were expressed in BL21(DE3) cells grown in Luria-Bertani (LB) medium in the presence of ampicillin (100 μg ml−1 ) at 37 ℃, and protein expression was induced at 18 ℃ with 0.4 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) at OD600 ≈ 0.5 for ~48 h. Cells were harvested at OD600 ≈ 1.5 and were suspended in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1% EDTA-free protease inhibitor cocktail (Sigma-Aldrich), and 20 mM imidazole.

Isolation:

Article Title: ATP Synthase C-Subunit-Deficient Mitochondria Have a Small Cyclosporine A-Sensitive Channel, but Lack the Permeability Transition Pore
Article Snippet: .. Mitoplast preparation Mitochondria from HAP1 and HAP1 – A12 cells were isolated by homogenization and differential centrifugation in mannitol-sucrose buffer (225 mM mannitol, 75 mM sucrose, 5 mM Tris-HCl, pH = 7.4) completed by 1 mM of PMSF (Thermo Fisher), EDTA-free protease inhibitor cocktail (Sigma Aldrich) and 1 mM of EGTA (Sigma Aldrich). ..

Transfection:

Article Title: ISWI chromatin remodellers sense nucleosome modifications to determine substrate preference
Article Snippet: In brief, BL21 Rosetta (DE3) cells were transfected with histone expression plasmids (pET, Novagen) and grown in LB medium at 37 °C until they reached an OD600 of 0.6. .. Cell pellets were resuspended in 10 ml of cold lysis buffer (50 mM Tris, 100 mM NaCl, 1 mM EDTA, 1 mM 2-mercaptoethanol, pH 7.6 at 4 °C) with cOmplete, EDTA-free Protease Inhibitor Cocktail (Sigma-Aldrich) per litre of culture and homogenized by passage several times through an 18-gauge needle.

Labeling:

Article Title: SIRT7 is an RNA-activated protein lysine deacylase
Article Snippet: Paragraph title: Global fluorescent labeling of fatty-acylated proteins in Sirt7 ctrl and KD cells ... The cell pellet was re-suspended in Buffer A (20 mM Tris, pH 8.0, 10 mM KCl, 1.5 mM MgCl2 , 1mM DTT, 5% glycerol, 0.1% NP-40) with EDTA-free protease inhibitor cocktail (Sigma) freshly added and incubated on ice for 5 mins.

Purification:

Article Title: The SMX DNA Repair Tri-nuclease
Article Snippet: Cells were resuspended in HisTRAP buffer (25 mM sodium phosphate pH 7.8, 500 mM NaCl, 10% glycerol, 0.05% NP-40, 1 mM DTT, 5 mM imidazole) supplemented with EDTA-Free Protease Inhibitor Cocktail (Sigma) and lysed by sonication on ice (5 × 15 s with 45 s incubations on ice in between) using a Soniprep 150 (MSE) at 50% maximum setting. .. The soluble extract was purified by immobilized metal affinity chromatography using a 5 mL HisTRAP FF column (GE Healthcare).

Article Title: USP48 restrains resection by site-specific cleavage of the BRCA1 ubiquitin mark from H2A
Article Snippet: Cells were infected at a density of 1 × 106 cells per ml and grown for 72 h. Cells were collected in lysis buffer (50 mM TRIS pH 8, 150 mM NaCl, 2 mM TCEP) and complete EDTA-free protease inhibitor (Sigma) was added. .. The sample was then purified on a Superdex 200 size exclusion column (GE Healthcare) in lysis buffer.

Article Title: ISWI chromatin remodellers sense nucleosome modifications to determine substrate preference
Article Snippet: Paragraph title: Production and purification of recombinant histones ... Cell pellets were resuspended in 10 ml of cold lysis buffer (50 mM Tris, 100 mM NaCl, 1 mM EDTA, 1 mM 2-mercaptoethanol, pH 7.6 at 4 °C) with cOmplete, EDTA-free Protease Inhibitor Cocktail (Sigma-Aldrich) per litre of culture and homogenized by passage several times through an 18-gauge needle.

Article Title: Expression and purification of the functional ectodomain of human anthrax toxin receptor 2 in E. coli Origami B cells with assistance of bacterial Trigger Factor
Article Snippet: Paragraph title: Preparative Expression and Purification of R318 ... Bacterial cells were harvested by centrifugation and the cell paste was re-suspended in Buffer A (20 mM TrisHCl pH 7.3, 300 mM NaCl, 10mM imidazole) with 1mg/ml of lysozyme and EDTA-free protease inhibitor cocktail (1 tablet/100 ml) (Sigma).

Article Title: Structures of chaperone-substrate complexes docked onto the export gate in a type III secretion system
Article Snippet: Unlabeled proteins were expressed in BL21(DE3) cells grown in Luria-Bertani (LB) medium in the presence of ampicillin (100 μg ml−1 ) at 37 ℃, and protein expression was induced at 18 ℃ with 0.4 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) at OD600 ≈ 0.5 for ~48 h. Cells were harvested at OD600 ≈ 1.5 and were suspended in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1% EDTA-free protease inhibitor cocktail (Sigma-Aldrich), and 20 mM imidazole. .. Cells were disrupted by a high-pressure homogenizer and centrifuged at 20,000 r.p.m. for 1 h. Proteins were purified using Ni Sepharose 6 Fast Flow resin (GE Healthcare), followed by tag removal by TEV protease at 4 ℃ for 12–20 h and gel filtration using Superdex 75 16/60.

Protein Purification:

Article Title: Visualizing conformational dynamics of proteins in solution and at the cell membrane
Article Snippet: Paragraph title: Protein purification and western blot analysis ... For fluorometry experiments, between 1 and 4 frozen pellets were thawed and resuspended in 0.5–0.8 mL Stabilization Buffer Tris (SBT) (in mM: KCl 70; MgCl2 1; Trizma Base 30; pH 7.4) supplemented with cOmplete mini EDTA-free protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO).

Protein Extraction:

Article Title: Targeting lonidamine to mitochondria mitigates lung tumorigenesis and brain metastasis
Article Snippet: .. Autophagy assessment in vivo by Western blot analysis Lung tissues harvested at necropsy were processed into protein lysates by homogenization (Bio-Gen PRO200, PRO Scientific Inc., Oxford, CT) and sonication (50 watt sonic dismembrator, Fisher Scientific, Hampton, NH) in T-PER® Tissue Protein Extraction Reagent (Thermo Fisher Scientific) with cOmplete™ EDTA-free protease inhibitor cocktail and PhosSTOP phosphatase inhibitors (Sigma-Aldrich) according to manufacturer’s instructions. .. Following centrifugation to remove insoluble material, protein levels in soluble lysates were quantified using the DC protein assay (Bio-Rad) and 15 µg/lane was loaded onto precast 4–20% Mini-Protean and Criterion TGX gels (Bio-Rad).

Affinity Chromatography:

Article Title: The SMX DNA Repair Tri-nuclease
Article Snippet: Cells were resuspended in HisTRAP buffer (25 mM sodium phosphate pH 7.8, 500 mM NaCl, 10% glycerol, 0.05% NP-40, 1 mM DTT, 5 mM imidazole) supplemented with EDTA-Free Protease Inhibitor Cocktail (Sigma) and lysed by sonication on ice (5 × 15 s with 45 s incubations on ice in between) using a Soniprep 150 (MSE) at 50% maximum setting. .. The soluble extract was purified by immobilized metal affinity chromatography using a 5 mL HisTRAP FF column (GE Healthcare).

Fast Protein Liquid Chromatography:

Article Title: Expression and purification of the functional ectodomain of human anthrax toxin receptor 2 in E. coli Origami B cells with assistance of bacterial Trigger Factor
Article Snippet: Bacterial cells were harvested by centrifugation and the cell paste was re-suspended in Buffer A (20 mM TrisHCl pH 7.3, 300 mM NaCl, 10mM imidazole) with 1mg/ml of lysozyme and EDTA-free protease inhibitor cocktail (1 tablet/100 ml) (Sigma). .. The soluble supernatant was passed through a Nickel-charged Sepharose column (50 ml, GE Healthcare), washed with 3 column volumes (CV) of Buffer A, and then eluted with a linear gradient of Buffer B (Buffer A + 500 mM imidazole) on an AKTA FPLC (GE Healthcare).

SDS Page:

Article Title: Gastrodin Inhibits Store-Operated Ca2+ Entry and Alleviates Cardiac Hypertrophy
Article Snippet: Western Blot Cellular and tissue proteins were extracted with lysis buffer, which contained 1% (vol/vol) Nonidet P-40, 150 mmol/L NaCl, 20 mmol/L Tris-HCl, pH 8.0, with the addition of Roche protease inhibitor cocktail tablets (Sigma–Aldrich, St. Louis, MO, USA, 04693132001). .. The proteins were separated in 12% SDS-PAGE and transferred to PVDF membranes.

Plasmid Preparation:

Article Title: Structures of chaperone-substrate complexes docked onto the export gate in a type III secretion system
Article Snippet: A 20-residue-long linker (VLFQGPSAGLVPRGSGGIEG) was selected from the pCold vector (Takara Bio). .. Unlabeled proteins were expressed in BL21(DE3) cells grown in Luria-Bertani (LB) medium in the presence of ampicillin (100 μg ml−1 ) at 37 ℃, and protein expression was induced at 18 ℃ with 0.4 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) at OD600 ≈ 0.5 for ~48 h. Cells were harvested at OD600 ≈ 1.5 and were suspended in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1% EDTA-free protease inhibitor cocktail (Sigma-Aldrich), and 20 mM imidazole.

Positron Emission Tomography:

Article Title: ISWI chromatin remodellers sense nucleosome modifications to determine substrate preference
Article Snippet: In brief, BL21 Rosetta (DE3) cells were transfected with histone expression plasmids (pET, Novagen) and grown in LB medium at 37 °C until they reached an OD600 of 0.6. .. Cell pellets were resuspended in 10 ml of cold lysis buffer (50 mM Tris, 100 mM NaCl, 1 mM EDTA, 1 mM 2-mercaptoethanol, pH 7.6 at 4 °C) with cOmplete, EDTA-free Protease Inhibitor Cocktail (Sigma-Aldrich) per litre of culture and homogenized by passage several times through an 18-gauge needle.

Homogenization:

Article Title: ATP Synthase C-Subunit-Deficient Mitochondria Have a Small Cyclosporine A-Sensitive Channel, but Lack the Permeability Transition Pore
Article Snippet: .. Mitoplast preparation Mitochondria from HAP1 and HAP1 – A12 cells were isolated by homogenization and differential centrifugation in mannitol-sucrose buffer (225 mM mannitol, 75 mM sucrose, 5 mM Tris-HCl, pH = 7.4) completed by 1 mM of PMSF (Thermo Fisher), EDTA-free protease inhibitor cocktail (Sigma Aldrich) and 1 mM of EGTA (Sigma Aldrich). ..

Article Title: Targeting lonidamine to mitochondria mitigates lung tumorigenesis and brain metastasis
Article Snippet: .. Autophagy assessment in vivo by Western blot analysis Lung tissues harvested at necropsy were processed into protein lysates by homogenization (Bio-Gen PRO200, PRO Scientific Inc., Oxford, CT) and sonication (50 watt sonic dismembrator, Fisher Scientific, Hampton, NH) in T-PER® Tissue Protein Extraction Reagent (Thermo Fisher Scientific) with cOmplete™ EDTA-free protease inhibitor cocktail and PhosSTOP phosphatase inhibitors (Sigma-Aldrich) according to manufacturer’s instructions. .. Following centrifugation to remove insoluble material, protein levels in soluble lysates were quantified using the DC protein assay (Bio-Rad) and 15 µg/lane was loaded onto precast 4–20% Mini-Protean and Criterion TGX gels (Bio-Rad).

Nuclear Magnetic Resonance:

Article Title: The SMX DNA Repair Tri-nuclease
Article Snippet: Peak fractions were pooled and dialyzed against NMR buffer (25 mM Na2 HPO4 , pH 7.0, 500 mM NaCl, 2 mM DTT, 2 mM benzamide, 0.5 mM PMSF). .. Cells were resuspended in HisTRAP buffer (25 mM sodium phosphate pH 7.8, 500 mM NaCl, 10% glycerol, 0.05% NP-40, 1 mM DTT, 5 mM imidazole) supplemented with EDTA-Free Protease Inhibitor Cocktail (Sigma) and lysed by sonication on ice (5 × 15 s with 45 s incubations on ice in between) using a Soniprep 150 (MSE) at 50% maximum setting.

Produced:

Article Title: ISWI chromatin remodellers sense nucleosome modifications to determine substrate preference
Article Snippet: Unmodified recombinant human histones (H2A, Uniprot ID: ; H2B, Uniprot ID: ; H3C96A, C110A, Uniprot ID: ; H4, Uniprot ID: ), histone variants, and histone mutants were produced in and purified from E. coli . .. Cell pellets were resuspended in 10 ml of cold lysis buffer (50 mM Tris, 100 mM NaCl, 1 mM EDTA, 1 mM 2-mercaptoethanol, pH 7.6 at 4 °C) with cOmplete, EDTA-free Protease Inhibitor Cocktail (Sigma-Aldrich) per litre of culture and homogenized by passage several times through an 18-gauge needle.

Article Title: New Use for CETSA: Monitoring Innate Immune Receptor Stability via Post-Translational Modification by OGT
Article Snippet: All antibodies were purchased from Cell Signaling Technology, except CTD110.6, which was produced and gifted by Core C4 (Department of Biological Chemistry, Johns Hopkins University), and Flag, which was purchased from Sigma-Aldrich. .. MDP was purchased from Bachem. iE-DAP was purchased from Invivogen. cOmplete EDTA-free protease inhibitor cocktail was purchased from Sigma-Aldrich.

Immunoprecipitation:

Article Title: Amino acids disrupt calcium-dependent adhesion of stratum corneum
Article Snippet: Paragraph title: Immunoprecipitation ... Tissues were washed with cold PBS, and tissue pellets were incubated in RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% NP-40, 1% Triton X-100) containing EDTA-free protease inhibitor cocktail (Sigma-Aldrich) for 15 min on ice.

Construct:

Article Title: Structures of chaperone-substrate complexes docked onto the export gate in a type III secretion system
Article Snippet: All mutants were constructed by site-directed mutagenesis using PfuTurbo high-fidelity DNA polymerase (Agilent). .. Unlabeled proteins were expressed in BL21(DE3) cells grown in Luria-Bertani (LB) medium in the presence of ampicillin (100 μg ml−1 ) at 37 ℃, and protein expression was induced at 18 ℃ with 0.4 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) at OD600 ≈ 0.5 for ~48 h. Cells were harvested at OD600 ≈ 1.5 and were suspended in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1% EDTA-free protease inhibitor cocktail (Sigma-Aldrich), and 20 mM imidazole.

Lysis:

Article Title: USP48 restrains resection by site-specific cleavage of the BRCA1 ubiquitin mark from H2A
Article Snippet: .. Cells were infected at a density of 1 × 106 cells per ml and grown for 72 h. Cells were collected in lysis buffer (50 mM TRIS pH 8, 150 mM NaCl, 2 mM TCEP) and complete EDTA-free protease inhibitor (Sigma) was added. .. Cells were lysed by sonication and sample was cleared by centrifugation at 21,000 × g for 30 min. Supernatant was loaded on His-affinity column (GE Healthcare) and column was washed with lysis buffer + 50 mM Imidazol.

Article Title: Gastrodin Inhibits Store-Operated Ca2+ Entry and Alleviates Cardiac Hypertrophy
Article Snippet: .. Western Blot Cellular and tissue proteins were extracted with lysis buffer, which contained 1% (vol/vol) Nonidet P-40, 150 mmol/L NaCl, 20 mmol/L Tris-HCl, pH 8.0, with the addition of Roche protease inhibitor cocktail tablets (Sigma–Aldrich, St. Louis, MO, USA, 04693132001). ..

Article Title: Amino acids disrupt calcium-dependent adhesion of stratum corneum
Article Snippet: Tissues were washed with cold PBS, and tissue pellets were incubated in RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% NP-40, 1% Triton X-100) containing EDTA-free protease inhibitor cocktail (Sigma-Aldrich) for 15 min on ice. .. Protein G sepharose 4B (Invitrogen) was then added and incubated for 4 h. Samples were washed four times with lysis buffer, boiled in 5× sample buffer containing β-mercaptoethanol, and subjected to electrophoresis on an 4–15% SDS-polyacrylamide gel (Bio-Rad).

Article Title: ISWI chromatin remodellers sense nucleosome modifications to determine substrate preference
Article Snippet: .. Cell pellets were resuspended in 10 ml of cold lysis buffer (50 mM Tris, 100 mM NaCl, 1 mM EDTA, 1 mM 2-mercaptoethanol, pH 7.6 at 4 °C) with cOmplete, EDTA-free Protease Inhibitor Cocktail (Sigma-Aldrich) per litre of culture and homogenized by passage several times through an 18-gauge needle. .. The inclusion body pellet was washed twice with cold lysis buffer containing 1% Triton-X 100 and once without detergent.

Article Title: USP48 restrains resection by site-specific cleavage of the BRCA1 ubiquitin mark from H2A
Article Snippet: .. Cells were collected in lysis buffer (50 mM TRIS pH 8, 100 mM NaCl, 1 mM β-mercaptoethanol) with Complete EDTA-free protease inhibitor (Sigma) and lysed by sonication. .. Lysate was cleared by spinning down at 21,000 × g and loaded on TALON beads (Clontech Laboratories, Inc.).

Article Title: Protective effect of stromal Dickkopf-3 in prostate cancer: opposing roles for TGFBI and ECM-1
Article Snippet: .. Cells were lysed on ice in RIPA lysis buffer (0.5% Na deoxycholate, 1% Triton X-100, 20 mM Tris pH 8.0, 0.1% SDS, 100 mM NaCl, 50 mM NaF, 1 mM EDTA), cOmplete™ EDTA-free Protease Inhibitor Cocktail (Sigma) and PhosSTOP Phosphatase Inhibitor Cocktail (Sigma)). .. Lysates were incubated for 15 min on ice, centrifuged at 15,000× g for 15 min at 4 °C and added to an equal volume of 2× Laemmli buffer.

Article Title: Structures of chaperone-substrate complexes docked onto the export gate in a type III secretion system
Article Snippet: .. Unlabeled proteins were expressed in BL21(DE3) cells grown in Luria-Bertani (LB) medium in the presence of ampicillin (100 μg ml−1 ) at 37 ℃, and protein expression was induced at 18 ℃ with 0.4 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) at OD600 ≈ 0.5 for ~48 h. Cells were harvested at OD600 ≈ 1.5 and were suspended in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1% EDTA-free protease inhibitor cocktail (Sigma-Aldrich), and 20 mM imidazole. .. Cells were disrupted by a high-pressure homogenizer and centrifuged at 20,000 r.p.m. for 1 h. Proteins were purified using Ni Sepharose 6 Fast Flow resin (GE Healthcare), followed by tag removal by TEV protease at 4 ℃ for 12–20 h and gel filtration using Superdex 75 16/60.

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  • 99
    Millipore edta free
    Differential binding of wild-type and mutant CovR. Biotinylated promoter regions of gbpC and SMU.1882 were immobilized on streptavidin biosensors and then exposed to 0.5 µM CovR, CovR D53A and CovR D53E proteins purified from E. coli as described in the text. The reactions were carried out in binding buffer [20 mM <t>Tris,</t> 100 mM NaCl, 0.01 mM DTT, 5 % glycerol (v/v), 1 mM <t>EDTA,</t> 0.01 mg ml −1 BSA, 5 mM MgCl 2 and 10 µg ml −1 poly (dI-dC) at pH 7.5] for a period of 5 min to allow association followed by 2 min exposure to binding buffer to allow dissociation.
    Edta Free, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 100 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/edta free/product/Millipore
    Average 99 stars, based on 100 article reviews
    Price from $9.99 to $1999.99
    edta free - by Bioz Stars, 2020-03
    99/100 stars
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    99
    Millipore v ethylenediaminetetraacetic acid free
    Differential binding of wild-type and mutant CovR. Biotinylated promoter regions of gbpC and SMU.1882 were immobilized on streptavidin biosensors and then exposed to 0.5 µM CovR, CovR D53A and CovR D53E proteins purified from E. coli as described in the text. The reactions were carried out in binding buffer [20 mM <t>Tris,</t> 100 mM NaCl, 0.01 mM DTT, 5 % glycerol (v/v), 1 mM <t>EDTA,</t> 0.01 mg ml −1 BSA, 5 mM MgCl 2 and 10 µg ml −1 poly (dI-dC) at pH 7.5] for a period of 5 min to allow association followed by 2 min exposure to binding buffer to allow dissociation.
    V Ethylenediaminetetraacetic Acid Free, supplied by Millipore, 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/v ethylenediaminetetraacetic acid free/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    v ethylenediaminetetraacetic acid free - by Bioz Stars, 2020-03
    99/100 stars
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    Differential binding of wild-type and mutant CovR. Biotinylated promoter regions of gbpC and SMU.1882 were immobilized on streptavidin biosensors and then exposed to 0.5 µM CovR, CovR D53A and CovR D53E proteins purified from E. coli as described in the text. The reactions were carried out in binding buffer [20 mM Tris, 100 mM NaCl, 0.01 mM DTT, 5 % glycerol (v/v), 1 mM EDTA, 0.01 mg ml −1 BSA, 5 mM MgCl 2 and 10 µg ml −1 poly (dI-dC) at pH 7.5] for a period of 5 min to allow association followed by 2 min exposure to binding buffer to allow dissociation.

    Journal: Microbiology

    Article Title: Role of CovR phosphorylation in gene transcription in Streptococcus mutans

    doi: 10.1099/mic.0.000641

    Figure Lengend Snippet: Differential binding of wild-type and mutant CovR. Biotinylated promoter regions of gbpC and SMU.1882 were immobilized on streptavidin biosensors and then exposed to 0.5 µM CovR, CovR D53A and CovR D53E proteins purified from E. coli as described in the text. The reactions were carried out in binding buffer [20 mM Tris, 100 mM NaCl, 0.01 mM DTT, 5 % glycerol (v/v), 1 mM EDTA, 0.01 mg ml −1 BSA, 5 mM MgCl 2 and 10 µg ml −1 poly (dI-dC) at pH 7.5] for a period of 5 min to allow association followed by 2 min exposure to binding buffer to allow dissociation.

    Article Snippet: The cells were first resuspended in 1.0 ml of cold 50 mM Tris pH 7.0 and 8 µl of Protease Inhibitor Cocktail Set III, EDTA-free (Calbiochem).

    Techniques: Binding Assay, Mutagenesis, Purification