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Millipore buffer
Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 256 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 99 stars, based on 256 article reviews
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Centrifugation:

Article Title: Protein-enriched outer membrane vesicles as a native platform for outer membrane protein studies
Article Snippet: Outer membranes were collected by centrifugation (100,000 xg for 1 h) and solubilized in buffer (20 mM Tris-HCl, 150 mM NaCl, 1% (w v–1 ) n-dodecyl-N,N -dimethylamine-N-oxide (LDAO, Anatrace), pH 8). .. Omps were then bound to Protino Ni-NTA agarose (Macherey-Nagel), washed with buffer (20 mM Tris-HCl, 150 mM NaCl, 0.5% (w v–1 ) LDAO, pH 8) and buffer (20 mM Tris-HCl, 150 mM NaCl, 0.1% (w v–1 ) LDAO, 10 mM imidazole (Sigma Aldrich), pH 8), and eluted with buffer (20 mM Tris-HCl, 150 mM NaCl, 0.1% (w v–1 ) LDAO, 500 mM imidazole, pH 8).

Amplification:

Article Title: Identification and Characterization of microRNAs during Retinoic Acid-Induced Regeneration of a Molluscan Central Nervous System
Article Snippet: Paragraph title: 4.9. LNA-FISH and Tyramide Signal Amplification ... Following these washes, cells and tissues were incubated in blocking buffer (3% bovine serum, 4× SSC, 0.1% Tween-20) for 30 min, then horseradish peroxidase (HRP) (Sigma) for 30 min at room temperature.

Article Title: Proteomics dataset containing proteins that obscure identification of TOPLESS interactors in Arabidopsis
Article Snippet: An internal control (UBC21) and the target cDNA was amplified over 28 cycles, were separated via electrophoresis, and imaged using the Gel Logic 200 (Kodak). .. Tissue samples were frozen in liquid nitrogen and ground with the 2000 Geno/Grinder®, and suspended in 200 μL ice-cold grinding buffer (50 mM Tris-Cl pH 7.5, 150 mM NaCl, 0.1% NP-40, 0.1% SDS, 25 mM β-ME, 1x plant protease inhibitor cocktail (Sigma-Aldrich®)).

Positive Control:

Article Title: Insights Into Dolphins' Immunology: Immuno-Phenotypic Study on Mediterranean and Atlantic Stranded Cetaceans
Article Snippet: The samples were diluted 1:1 in 2x Laemmli sample buffer (Sigma–Aldrich, St. Louis, MO, USA), boiled for 5 min at 95°C and separated by 12% SDS–PAGE in a mini-gel apparatus (Hoefer SE 260, GE Healthcare, UK) under denaturing and reducing conditions in according to Laemmli protocol ( ). .. Homogenate of human tonsil was used as a positive control to test the binding with specific antibodies.

Blocking Assay:

Article Title: Identification and Characterization of microRNAs during Retinoic Acid-Induced Regeneration of a Molluscan Central Nervous System
Article Snippet: .. Following these washes, cells and tissues were incubated in blocking buffer (3% bovine serum, 4× SSC, 0.1% Tween-20) for 30 min, then horseradish peroxidase (HRP) (Sigma) for 30 min at room temperature. .. Samples were next washed in TNT buffer (0.1 M Tris HCl, 0.15 M NaCl, 0.05% Tween-20), then incubated in tyramide for 1 h at room temperature.

Article Title: Aminosalicylic acid reduces ER stress and Schwann cell death induced by MPZ mutations
Article Snippet: .. The membranes were then stained with 0.1% Ponceau S solution (Sigma-Aldrich) to ensure equal loading of the samples, and non-specific binding was blocked with a blocking buffer (Casein blocking buffer 10X; Sigma-Aldrich) for 1 h at room temperature. .. The membranes were incubated overnight at 4°C with the following primary antibodies: Anti-Myc-tag (1:2,000; ab9106, Abcam), anti-binding immunoglobulin protein (BiP, #3177), anti-C/EBP-homologous protein (CHOP, #5554), anti-cleaved caspase-3 (Asp175, #9661), anti-phospho-AKT (ser473, #9271), anti-phospho-SAPK/JNK (Thr183/Tyr185, #9251) (all 1:1,000) (all from Cell Signaling Technology) and anti-β-actin (A2228, Sigma-Aldrich).

Article Title: Ubiquitin-mediated proteasome degradation regulates optic fissure fusion
Article Snippet: A fourth combination repeated Siah1:MYC pCIG2 (2 µg)+Nlz2:FLAG pCIG2 (1 µg) along with a treatment using 10 mM of MG132 in the last 4 h. Protein extraction was performed by adding Laemmli sample buffer (Sigma-Aldrich) and boiling the samples at 95°C for 10 min. .. The membrane was blocked overnight at 4°C using Odyssey blocking buffer (Li-COR).

Electrophoresis:

Article Title: Insights Into Dolphins' Immunology: Immuno-Phenotypic Study on Mediterranean and Atlantic Stranded Cetaceans
Article Snippet: The samples were diluted 1:1 in 2x Laemmli sample buffer (Sigma–Aldrich, St. Louis, MO, USA), boiled for 5 min at 95°C and separated by 12% SDS–PAGE in a mini-gel apparatus (Hoefer SE 260, GE Healthcare, UK) under denaturing and reducing conditions in according to Laemmli protocol ( ). .. Following electrophoresis, gels were blotted (350 V, 1 h, 4°C) onto nitrocellulose membranes (0.45 μm; GE Healthcare, UK) in Laemmli transfer buffer (25 mM TRIS-base, 192 mM Glycine and 20% Methanol, pH 8.3) using a trans blot apparatus (Elettrofor, Rovigo, Italy).

Article Title: Perturbations of the ZED1 pseudokinase activate plant immunity
Article Snippet: Powdered frozen plant tissue was then suspended in 100 μL of Grinding Buffer (40 mM Tris pH = 7.5, 150 mM NaCl, 1 mM EDTA, 5 mM DTT, 1% Triton X-100, 0.1% sodium dodecyl sulfate) supplemented with a 1:500 dilution of plant protease inhibitor cocktail P9599 (Sigma). .. 15 uL from each of these extracts was then resolved by electrophoresis through 10% polyacrylamide SDS-PAGE gels prior to protein transfer to nitrocellulose membranes.

Article Title: Proteomics dataset containing proteins that obscure identification of TOPLESS interactors in Arabidopsis
Article Snippet: An internal control (UBC21) and the target cDNA was amplified over 28 cycles, were separated via electrophoresis, and imaged using the Gel Logic 200 (Kodak). .. Tissue samples were frozen in liquid nitrogen and ground with the 2000 Geno/Grinder®, and suspended in 200 μL ice-cold grinding buffer (50 mM Tris-Cl pH 7.5, 150 mM NaCl, 0.1% NP-40, 0.1% SDS, 25 mM β-ME, 1x plant protease inhibitor cocktail (Sigma-Aldrich®)).

Incubation:

Article Title: Identification and Characterization of microRNAs during Retinoic Acid-Induced Regeneration of a Molluscan Central Nervous System
Article Snippet: .. Following these washes, cells and tissues were incubated in blocking buffer (3% bovine serum, 4× SSC, 0.1% Tween-20) for 30 min, then horseradish peroxidase (HRP) (Sigma) for 30 min at room temperature. .. Samples were next washed in TNT buffer (0.1 M Tris HCl, 0.15 M NaCl, 0.05% Tween-20), then incubated in tyramide for 1 h at room temperature.

Article Title: Aminosalicylic acid reduces ER stress and Schwann cell death induced by MPZ mutations
Article Snippet: The membranes were then stained with 0.1% Ponceau S solution (Sigma-Aldrich) to ensure equal loading of the samples, and non-specific binding was blocked with a blocking buffer (Casein blocking buffer 10X; Sigma-Aldrich) for 1 h at room temperature. .. The membranes were incubated overnight at 4°C with the following primary antibodies: Anti-Myc-tag (1:2,000; ab9106, Abcam), anti-binding immunoglobulin protein (BiP, #3177), anti-C/EBP-homologous protein (CHOP, #5554), anti-cleaved caspase-3 (Asp175, #9661), anti-phospho-AKT (ser473, #9271), anti-phospho-SAPK/JNK (Thr183/Tyr185, #9251) (all 1:1,000) (all from Cell Signaling Technology) and anti-β-actin (A2228, Sigma-Aldrich).

Article Title: Ubiquitin-mediated proteasome degradation regulates optic fissure fusion
Article Snippet: A fourth combination repeated Siah1:MYC pCIG2 (2 µg)+Nlz2:FLAG pCIG2 (1 µg) along with a treatment using 10 mM of MG132 in the last 4 h. Protein extraction was performed by adding Laemmli sample buffer (Sigma-Aldrich) and boiling the samples at 95°C for 10 min. .. The membrane was incubated with rabbit anti-MYC (Sigma-Aldrich, 1:750) and mouse anti-FLAG (Sigma-Aldrich, 1:750) in 50% blocking buffer/PBS for 3 h at room temperature and immediately washed in five times PBST for 5 min. For detection of HA, rabbit anti-HA (Santa Cruz 1:500) was used.

Article Title: Tomato SlSAP3, a member of the stress‐associated protein family, is a positive regulator of immunity against Pseudomonas syringae pv. tomato DC3000
Article Snippet: .. Briefly, reactions (30 μL) contained 5 μg ubiquitin (Boston Biochem, Cambridge, MA, USA), 110 ng E1 (Merck Millipore, Darmstadt, Germany), 100 ng human recombinant UbcH2 (Abcam, Cambridge, UK), and purified 4 μg GST‐SlSAP3 in buffer (20 mM MOPS, pH 7.2, 100 mM KCl, 5 mM MgCl2 , 5 mM ATP and 10 mM DTT) and were incubated at 30 °C for 3 h. Reactions were stopped by adding SDS sample buffer and analysed by SDS‐PAGE. followed by immunoblotting using anti‐ubiquitin antibody (CalBiochem, La Jolla, CA, USA). .. Chemiluminescence signal was detected with SuperSignal West Pico PLUS Chemiluminescent Substrate kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's recommendations.

Activity Assay:

Article Title: Tomato SlSAP3, a member of the stress‐associated protein family, is a positive regulator of immunity against Pseudomonas syringae pv. tomato DC3000
Article Snippet: Paragraph title: Purification of recombinant GST‐SlSAP3 protein and ubiquitin E3 ligase activity assay ... Briefly, reactions (30 μL) contained 5 μg ubiquitin (Boston Biochem, Cambridge, MA, USA), 110 ng E1 (Merck Millipore, Darmstadt, Germany), 100 ng human recombinant UbcH2 (Abcam, Cambridge, UK), and purified 4 μg GST‐SlSAP3 in buffer (20 mM MOPS, pH 7.2, 100 mM KCl, 5 mM MgCl2 , 5 mM ATP and 10 mM DTT) and were incubated at 30 °C for 3 h. Reactions were stopped by adding SDS sample buffer and analysed by SDS‐PAGE. followed by immunoblotting using anti‐ubiquitin antibody (CalBiochem, La Jolla, CA, USA).

Expressing:

Article Title: Protein-enriched outer membrane vesicles as a native platform for outer membrane protein studies
Article Snippet: Briefly, to express Omps under leaky expression conditions in BL21(DE3)omp8 bacterial cultures were inoculated at a ratio of 1:100 from overnight cultures and grown in 2 L LB medium (Difco), supplemented with 100 µg mL–1 ampicillin (Sigma Aldrich) in 5 L Erlenmeyer flasks at 20 °C for 24 h. Cells were harvested (5000xg for 12 min), resuspended in buffer (20 mM Tris-HCl, 100 mM NaCl, pH 8) and broken by sonication. .. Omps were then bound to Protino Ni-NTA agarose (Macherey-Nagel), washed with buffer (20 mM Tris-HCl, 150 mM NaCl, 0.5% (w v–1 ) LDAO, pH 8) and buffer (20 mM Tris-HCl, 150 mM NaCl, 0.1% (w v–1 ) LDAO, 10 mM imidazole (Sigma Aldrich), pH 8), and eluted with buffer (20 mM Tris-HCl, 150 mM NaCl, 0.1% (w v–1 ) LDAO, 500 mM imidazole, pH 8).

Article Title: Perturbations of the ZED1 pseudokinase activate plant immunity
Article Snippet: Expression of transgenes in N . benthamiana was induced by spraying infiltrated plants with a solution of 20 μM dexamethasone in ddH2 0 with 0.01% Silwet L-77. .. Powdered frozen plant tissue was then suspended in 100 μL of Grinding Buffer (40 mM Tris pH = 7.5, 150 mM NaCl, 1 mM EDTA, 5 mM DTT, 1% Triton X-100, 0.1% sodium dodecyl sulfate) supplemented with a 1:500 dilution of plant protease inhibitor cocktail P9599 (Sigma).

Article Title: Aminosalicylic acid reduces ER stress and Schwann cell death induced by MPZ mutations
Article Snippet: Western blot analysis The expression of MPZ proteins and ER stress markers was confirmed using a standard western blot analysis. .. The membranes were then stained with 0.1% Ponceau S solution (Sigma-Aldrich) to ensure equal loading of the samples, and non-specific binding was blocked with a blocking buffer (Casein blocking buffer 10X; Sigma-Aldrich) for 1 h at room temperature.

Article Title: Tomato SlSAP3, a member of the stress‐associated protein family, is a positive regulator of immunity against Pseudomonas syringae pv. tomato DC3000
Article Snippet: The resulting plasmid was introduced into the E. coli strain Rosetta DE3 and expression of GST‐SlSAP3 fusion was induced by adding of 1 mM isopropyl‐D‐thiogalactoside (IPTG) at 20 °C overnight. .. Briefly, reactions (30 μL) contained 5 μg ubiquitin (Boston Biochem, Cambridge, MA, USA), 110 ng E1 (Merck Millipore, Darmstadt, Germany), 100 ng human recombinant UbcH2 (Abcam, Cambridge, UK), and purified 4 μg GST‐SlSAP3 in buffer (20 mM MOPS, pH 7.2, 100 mM KCl, 5 mM MgCl2 , 5 mM ATP and 10 mM DTT) and were incubated at 30 °C for 3 h. Reactions were stopped by adding SDS sample buffer and analysed by SDS‐PAGE. followed by immunoblotting using anti‐ubiquitin antibody (CalBiochem, La Jolla, CA, USA).

Article Title: Proteomics dataset containing proteins that obscure identification of TOPLESS interactors in Arabidopsis
Article Snippet: One hundred milligrams of leaves were collected from eight kanamycin-resistant T2 seedlings to analyze TPL-GS-TAG protein expression. .. Tissue samples were frozen in liquid nitrogen and ground with the 2000 Geno/Grinder®, and suspended in 200 μL ice-cold grinding buffer (50 mM Tris-Cl pH 7.5, 150 mM NaCl, 0.1% NP-40, 0.1% SDS, 25 mM β-ME, 1x plant protease inhibitor cocktail (Sigma-Aldrich®)).

BIA-KA:

Article Title: Insights Into Dolphins' Immunology: Immuno-Phenotypic Study on Mediterranean and Atlantic Stranded Cetaceans
Article Snippet: Total protein concentration was determined using BCA Protein Assay Kit (Pierce Biotechnology, USA). .. The samples were diluted 1:1 in 2x Laemmli sample buffer (Sigma–Aldrich, St. Louis, MO, USA), boiled for 5 min at 95°C and separated by 12% SDS–PAGE in a mini-gel apparatus (Hoefer SE 260, GE Healthcare, UK) under denaturing and reducing conditions in according to Laemmli protocol ( ).

Article Title: Aminosalicylic acid reduces ER stress and Schwann cell death induced by MPZ mutations
Article Snippet: The cell lysates were centrifuged at 13,000 × g for 15 min at 4°C, and the supernatants were used for quantification using bicinchoninic acid (BCA) method. .. The membranes were then stained with 0.1% Ponceau S solution (Sigma-Aldrich) to ensure equal loading of the samples, and non-specific binding was blocked with a blocking buffer (Casein blocking buffer 10X; Sigma-Aldrich) for 1 h at room temperature.

Modification:

Article Title: A unique intracellular tyrosine in neuroligin-1 regulates AMPA receptor recruitment during synapse differentiation and potentiation
Article Snippet: GST pull-down COS-7 cells (ATCC) transfected with gephyrin-Venus were washed with cold PBS, then lysed on ice by adding the following buffer (150 mM NaCl, 50 mM Tris, 1% Triton X-100, 1 mM EDTA, protease inhibitor cocktail III (Calbiochem)) to each well (400 µl/ 6-well plate, 3 wells per condition). .. In parallel, Pierce® Glutathione Magnetic Beads were equilibrated in a modified PBS solution (PBS pH 7.4, 0.01% BSA, 0.01% Tween-20) and incubated with either GST, GST-Nlg1, GST-Nlg1-Y782A, or GST-Nlg1-Y782F (0.64 nmol/200 µl modified PBS) for 20 min at RT on a shaker.

Article Title: Identification and Characterization of microRNAs during Retinoic Acid-Induced Regeneration of a Molluscan Central Nervous System
Article Snippet: For staining procedures, CNS tissue sections and cultured cells underwent the same procedures modified from Lu and Tsourkas [ ]. .. Following these washes, cells and tissues were incubated in blocking buffer (3% bovine serum, 4× SSC, 0.1% Tween-20) for 30 min, then horseradish peroxidase (HRP) (Sigma) for 30 min at room temperature.

Western Blot:

Article Title: Insights Into Dolphins' Immunology: Immuno-Phenotypic Study on Mediterranean and Atlantic Stranded Cetaceans
Article Snippet: Paragraph title: Western Blotting Analysis ... The samples were diluted 1:1 in 2x Laemmli sample buffer (Sigma–Aldrich, St. Louis, MO, USA), boiled for 5 min at 95°C and separated by 12% SDS–PAGE in a mini-gel apparatus (Hoefer SE 260, GE Healthcare, UK) under denaturing and reducing conditions in according to Laemmli protocol ( ).

Article Title: Aminosalicylic acid reduces ER stress and Schwann cell death induced by MPZ mutations
Article Snippet: Paragraph title: Western blot analysis ... The membranes were then stained with 0.1% Ponceau S solution (Sigma-Aldrich) to ensure equal loading of the samples, and non-specific binding was blocked with a blocking buffer (Casein blocking buffer 10X; Sigma-Aldrich) for 1 h at room temperature.

Article Title: Ubiquitin-mediated proteasome degradation regulates optic fissure fusion
Article Snippet: Paragraph title: Transfections and western blotting ... A fourth combination repeated Siah1:MYC pCIG2 (2 µg)+Nlz2:FLAG pCIG2 (1 µg) along with a treatment using 10 mM of MG132 in the last 4 h. Protein extraction was performed by adding Laemmli sample buffer (Sigma-Aldrich) and boiling the samples at 95°C for 10 min.

Transformation Assay:

Article Title: Perturbations of the ZED1 pseudokinase activate plant immunity
Article Snippet: For each transformation of interest, tissue was harvested by punching three circular leaf cores 5 mm in diameter. .. Powdered frozen plant tissue was then suspended in 100 μL of Grinding Buffer (40 mM Tris pH = 7.5, 150 mM NaCl, 1 mM EDTA, 5 mM DTT, 1% Triton X-100, 0.1% sodium dodecyl sulfate) supplemented with a 1:500 dilution of plant protease inhibitor cocktail P9599 (Sigma).

Hybridization:

Article Title: Identification and Characterization of microRNAs during Retinoic Acid-Induced Regeneration of a Molluscan Central Nervous System
Article Snippet: The next day, tissue sections and cells were incubated in hybridization buffer (25% formamide (Sigma), 0.05M EDTA (Sigma), 4× saline-sodium citrate (SSC) buffer (Sigma), 10% dextran sulfate (Sigma), 1× Denhart’s solution (Sigma), 0.5 mg/mL E. coli tRNA (Sigma), 20 mM ribonucleoside vanadyl complexes (Sigma), 9.2 mM citric acid (Sigma)) at 58 °C (for miR-124 and scrambled probe) or 52 °C (for miR-133 ) for 2 h, then hybridized in 10 nM of probe (Exiqon, Woburn, MA, USA) for 1 h at the same temperature. .. Following these washes, cells and tissues were incubated in blocking buffer (3% bovine serum, 4× SSC, 0.1% Tween-20) for 30 min, then horseradish peroxidase (HRP) (Sigma) for 30 min at room temperature.

Transfection:

Article Title: A unique intracellular tyrosine in neuroligin-1 regulates AMPA receptor recruitment during synapse differentiation and potentiation
Article Snippet: .. GST pull-down COS-7 cells (ATCC) transfected with gephyrin-Venus were washed with cold PBS, then lysed on ice by adding the following buffer (150 mM NaCl, 50 mM Tris, 1% Triton X-100, 1 mM EDTA, protease inhibitor cocktail III (Calbiochem)) to each well (400 µl/ 6-well plate, 3 wells per condition). ..

Article Title: Ubiquitin-mediated proteasome degradation regulates optic fissure fusion
Article Snippet: Paragraph title: Transfections and western blotting ... A fourth combination repeated Siah1:MYC pCIG2 (2 µg)+Nlz2:FLAG pCIG2 (1 µg) along with a treatment using 10 mM of MG132 in the last 4 h. Protein extraction was performed by adding Laemmli sample buffer (Sigma-Aldrich) and boiling the samples at 95°C for 10 min.

Protease Inhibitor:

Article Title: A unique intracellular tyrosine in neuroligin-1 regulates AMPA receptor recruitment during synapse differentiation and potentiation
Article Snippet: .. GST pull-down COS-7 cells (ATCC) transfected with gephyrin-Venus were washed with cold PBS, then lysed on ice by adding the following buffer (150 mM NaCl, 50 mM Tris, 1% Triton X-100, 1 mM EDTA, protease inhibitor cocktail III (Calbiochem)) to each well (400 µl/ 6-well plate, 3 wells per condition). ..

Article Title: Perturbations of the ZED1 pseudokinase activate plant immunity
Article Snippet: .. Powdered frozen plant tissue was then suspended in 100 μL of Grinding Buffer (40 mM Tris pH = 7.5, 150 mM NaCl, 1 mM EDTA, 5 mM DTT, 1% Triton X-100, 0.1% sodium dodecyl sulfate) supplemented with a 1:500 dilution of plant protease inhibitor cocktail P9599 (Sigma). .. 15 uL from each of these extracts was then resolved by electrophoresis through 10% polyacrylamide SDS-PAGE gels prior to protein transfer to nitrocellulose membranes.

Article Title: Proteomics dataset containing proteins that obscure identification of TOPLESS interactors in Arabidopsis
Article Snippet: .. Tissue samples were frozen in liquid nitrogen and ground with the 2000 Geno/Grinder®, and suspended in 200 μL ice-cold grinding buffer (50 mM Tris-Cl pH 7.5, 150 mM NaCl, 0.1% NP-40, 0.1% SDS, 25 mM β-ME, 1x plant protease inhibitor cocktail (Sigma-Aldrich®)). ..

Buffer Exchange:

Article Title: Protein-enriched outer membrane vesicles as a native platform for outer membrane protein studies
Article Snippet: Omps were then bound to Protino Ni-NTA agarose (Macherey-Nagel), washed with buffer (20 mM Tris-HCl, 150 mM NaCl, 0.5% (w v–1 ) LDAO, pH 8) and buffer (20 mM Tris-HCl, 150 mM NaCl, 0.1% (w v–1 ) LDAO, 10 mM imidazole (Sigma Aldrich), pH 8), and eluted with buffer (20 mM Tris-HCl, 150 mM NaCl, 0.1% (w v–1 ) LDAO, 500 mM imidazole, pH 8). .. Omps were reconstituted into E. coli polar lipid extract (Avanti polar Lipids) at a lipid to protein ratio of 0.5 (w w–1 ) by dialysis driven detergent removal against buffer (20 mM Tris-HCl, 150 mM NaCl, 0.01% (w v–1 ) NaN3 , pH 8) for 5 days at 30 °C with daily buffer exchange.

Cell Culture:

Article Title: Identification and Characterization of microRNAs during Retinoic Acid-Induced Regeneration of a Molluscan Central Nervous System
Article Snippet: For staining procedures, CNS tissue sections and cultured cells underwent the same procedures modified from Lu and Tsourkas [ ]. .. Following these washes, cells and tissues were incubated in blocking buffer (3% bovine serum, 4× SSC, 0.1% Tween-20) for 30 min, then horseradish peroxidase (HRP) (Sigma) for 30 min at room temperature.

other:

Article Title: Potent Anti‐Inflammatory and Pro‐Resolving Effects of Anabasum in a Human Model of Self‐Resolving Acute Inflammation
Article Snippet: For use with this kit, the cell‐free blister exudate was first treated, as recommended previously for human biological matrices, by diluting 1:20 in 0.1% tween 80 buffer (Sigma, St. Louis, MO) to a final volume of 100 μL, followed by heating at 70°C for 15 min.

Protein Concentration:

Article Title: Insights Into Dolphins' Immunology: Immuno-Phenotypic Study on Mediterranean and Atlantic Stranded Cetaceans
Article Snippet: Total protein concentration was determined using BCA Protein Assay Kit (Pierce Biotechnology, USA). .. The samples were diluted 1:1 in 2x Laemmli sample buffer (Sigma–Aldrich, St. Louis, MO, USA), boiled for 5 min at 95°C and separated by 12% SDS–PAGE in a mini-gel apparatus (Hoefer SE 260, GE Healthcare, UK) under denaturing and reducing conditions in according to Laemmli protocol ( ).

Article Title: Protein-enriched outer membrane vesicles as a native platform for outer membrane protein studies
Article Snippet: Omps were then bound to Protino Ni-NTA agarose (Macherey-Nagel), washed with buffer (20 mM Tris-HCl, 150 mM NaCl, 0.5% (w v–1 ) LDAO, pH 8) and buffer (20 mM Tris-HCl, 150 mM NaCl, 0.1% (w v–1 ) LDAO, 10 mM imidazole (Sigma Aldrich), pH 8), and eluted with buffer (20 mM Tris-HCl, 150 mM NaCl, 0.1% (w v–1 ) LDAO, 500 mM imidazole, pH 8). .. Elution buffer was exchanged to buffer (20 mM Tris-HCl, 150 mM NaCl, 0.1% (w v–1 ) LDAO, pH 8) using PD-10 desalting columns (GE Healthcare) and the protein concentration was adjusted to 1 mg mL–1 .

Article Title: Tomato SlSAP3, a member of the stress‐associated protein family, is a positive regulator of immunity against Pseudomonas syringae pv. tomato DC3000
Article Snippet: Protein concentration was determined using Bio‐Rad protein assay kit (Bio‐Rad, CA, USA) following the recommended method. .. Briefly, reactions (30 μL) contained 5 μg ubiquitin (Boston Biochem, Cambridge, MA, USA), 110 ng E1 (Merck Millipore, Darmstadt, Germany), 100 ng human recombinant UbcH2 (Abcam, Cambridge, UK), and purified 4 μg GST‐SlSAP3 in buffer (20 mM MOPS, pH 7.2, 100 mM KCl, 5 mM MgCl2 , 5 mM ATP and 10 mM DTT) and were incubated at 30 °C for 3 h. Reactions were stopped by adding SDS sample buffer and analysed by SDS‐PAGE. followed by immunoblotting using anti‐ubiquitin antibody (CalBiochem, La Jolla, CA, USA).

Reverse Transcription Polymerase Chain Reaction:

Article Title: Proteomics dataset containing proteins that obscure identification of TOPLESS interactors in Arabidopsis
Article Snippet: Ten nanograms of total RNA was converted to cDNA using the SuperScript™ III One-Step RT-PCR System with Platinum™ Taq (Invitrogen™). .. Tissue samples were frozen in liquid nitrogen and ground with the 2000 Geno/Grinder®, and suspended in 200 μL ice-cold grinding buffer (50 mM Tris-Cl pH 7.5, 150 mM NaCl, 0.1% NP-40, 0.1% SDS, 25 mM β-ME, 1x plant protease inhibitor cocktail (Sigma-Aldrich®)).

Sonication:

Article Title: Protein-enriched outer membrane vesicles as a native platform for outer membrane protein studies
Article Snippet: Briefly, to express Omps under leaky expression conditions in BL21(DE3)omp8 bacterial cultures were inoculated at a ratio of 1:100 from overnight cultures and grown in 2 L LB medium (Difco), supplemented with 100 µg mL–1 ampicillin (Sigma Aldrich) in 5 L Erlenmeyer flasks at 20 °C for 24 h. Cells were harvested (5000xg for 12 min), resuspended in buffer (20 mM Tris-HCl, 100 mM NaCl, pH 8) and broken by sonication. .. Omps were then bound to Protino Ni-NTA agarose (Macherey-Nagel), washed with buffer (20 mM Tris-HCl, 150 mM NaCl, 0.5% (w v–1 ) LDAO, pH 8) and buffer (20 mM Tris-HCl, 150 mM NaCl, 0.1% (w v–1 ) LDAO, 10 mM imidazole (Sigma Aldrich), pH 8), and eluted with buffer (20 mM Tris-HCl, 150 mM NaCl, 0.1% (w v–1 ) LDAO, 500 mM imidazole, pH 8).

Binding Assay:

Article Title: Insights Into Dolphins' Immunology: Immuno-Phenotypic Study on Mediterranean and Atlantic Stranded Cetaceans
Article Snippet: The samples were diluted 1:1 in 2x Laemmli sample buffer (Sigma–Aldrich, St. Louis, MO, USA), boiled for 5 min at 95°C and separated by 12% SDS–PAGE in a mini-gel apparatus (Hoefer SE 260, GE Healthcare, UK) under denaturing and reducing conditions in according to Laemmli protocol ( ). .. Homogenate of human tonsil was used as a positive control to test the binding with specific antibodies.

Article Title: Aminosalicylic acid reduces ER stress and Schwann cell death induced by MPZ mutations
Article Snippet: .. The membranes were then stained with 0.1% Ponceau S solution (Sigma-Aldrich) to ensure equal loading of the samples, and non-specific binding was blocked with a blocking buffer (Casein blocking buffer 10X; Sigma-Aldrich) for 1 h at room temperature. .. The membranes were incubated overnight at 4°C with the following primary antibodies: Anti-Myc-tag (1:2,000; ab9106, Abcam), anti-binding immunoglobulin protein (BiP, #3177), anti-C/EBP-homologous protein (CHOP, #5554), anti-cleaved caspase-3 (Asp175, #9661), anti-phospho-AKT (ser473, #9271), anti-phospho-SAPK/JNK (Thr183/Tyr185, #9251) (all 1:1,000) (all from Cell Signaling Technology) and anti-β-actin (A2228, Sigma-Aldrich).

Magnetic Beads:

Article Title: A unique intracellular tyrosine in neuroligin-1 regulates AMPA receptor recruitment during synapse differentiation and potentiation
Article Snippet: GST pull-down COS-7 cells (ATCC) transfected with gephyrin-Venus were washed with cold PBS, then lysed on ice by adding the following buffer (150 mM NaCl, 50 mM Tris, 1% Triton X-100, 1 mM EDTA, protease inhibitor cocktail III (Calbiochem)) to each well (400 µl/ 6-well plate, 3 wells per condition). .. In parallel, Pierce® Glutathione Magnetic Beads were equilibrated in a modified PBS solution (PBS pH 7.4, 0.01% BSA, 0.01% Tween-20) and incubated with either GST, GST-Nlg1, GST-Nlg1-Y782A, or GST-Nlg1-Y782F (0.64 nmol/200 µl modified PBS) for 20 min at RT on a shaker.

Article Title: Conserved residues in the wheat (Triticum aestivum) NAM-A1 NAC domain are required for protein binding and when mutated lead to delayed peduncle and flag leaf senescence
Article Snippet: Protein was extracted in a standard buffer (100 mM Tris-HCl, pH 7.5; 150 mM NaCl; 0.1% (v/v) Triton x-100; 1% (w/v) PVPP; 5 mM EDTA; 10% glycerol; 2 mM DTT; 1% protease inhibitors (Sigma)). .. 1 mL of protein extraction was applied to M5 anti-FLAG magnetic beads (Sigma).

Microscopy:

Article Title: Identification and Characterization of microRNAs during Retinoic Acid-Induced Regeneration of a Molluscan Central Nervous System
Article Snippet: Following these washes, cells and tissues were incubated in blocking buffer (3% bovine serum, 4× SSC, 0.1% Tween-20) for 30 min, then horseradish peroxidase (HRP) (Sigma) for 30 min at room temperature. .. Following a final series of washes in TNT buffer, all samples were mounted and imaged using a Carl Zeiss Axio Observer.Z1 inverted light/epifluorescence microscope, with ApoTome.2 optical sectioning (Carl Zeiss Canada Ltd., North York, ON, Canada).

Purification:

Article Title: A unique intracellular tyrosine in neuroligin-1 regulates AMPA receptor recruitment during synapse differentiation and potentiation
Article Snippet: GST pull-down COS-7 cells (ATCC) transfected with gephyrin-Venus were washed with cold PBS, then lysed on ice by adding the following buffer (150 mM NaCl, 50 mM Tris, 1% Triton X-100, 1 mM EDTA, protease inhibitor cocktail III (Calbiochem)) to each well (400 µl/ 6-well plate, 3 wells per condition). .. After washing, beads were incubated for another 20 min with the lysate containing Gephyrin-Venus (200 µl/tube), or with purified PSD-95-mCherry (0.01 nmol/100 µL) at RT on a shaker.

Article Title: Tomato SlSAP3, a member of the stress‐associated protein family, is a positive regulator of immunity against Pseudomonas syringae pv. tomato DC3000
Article Snippet: .. Briefly, reactions (30 μL) contained 5 μg ubiquitin (Boston Biochem, Cambridge, MA, USA), 110 ng E1 (Merck Millipore, Darmstadt, Germany), 100 ng human recombinant UbcH2 (Abcam, Cambridge, UK), and purified 4 μg GST‐SlSAP3 in buffer (20 mM MOPS, pH 7.2, 100 mM KCl, 5 mM MgCl2 , 5 mM ATP and 10 mM DTT) and were incubated at 30 °C for 3 h. Reactions were stopped by adding SDS sample buffer and analysed by SDS‐PAGE. followed by immunoblotting using anti‐ubiquitin antibody (CalBiochem, La Jolla, CA, USA). .. Chemiluminescence signal was detected with SuperSignal West Pico PLUS Chemiluminescent Substrate kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's recommendations.

Protein Extraction:

Article Title: Ubiquitin-mediated proteasome degradation regulates optic fissure fusion
Article Snippet: .. A fourth combination repeated Siah1:MYC pCIG2 (2 µg)+Nlz2:FLAG pCIG2 (1 µg) along with a treatment using 10 mM of MG132 in the last 4 h. Protein extraction was performed by adding Laemmli sample buffer (Sigma-Aldrich) and boiling the samples at 95°C for 10 min. .. Equal volume for each transfection was load on 10% SDS-PAGE and run for 90 min at 120 V. Then, they were transfer to a PVDF membrane for 90 min at 400 mA.

Article Title: Conserved residues in the wheat (Triticum aestivum) NAM-A1 NAC domain are required for protein binding and when mutated lead to delayed peduncle and flag leaf senescence
Article Snippet: Protein was extracted in a standard buffer (100 mM Tris-HCl, pH 7.5; 150 mM NaCl; 0.1% (v/v) Triton x-100; 1% (w/v) PVPP; 5 mM EDTA; 10% glycerol; 2 mM DTT; 1% protease inhibitors (Sigma)). .. 1 mL of protein extraction was applied to M5 anti-FLAG magnetic beads (Sigma).

Selection:

Article Title: Proteomics dataset containing proteins that obscure identification of TOPLESS interactors in Arabidopsis
Article Snippet: Paragraph title: Selection of plants ... Tissue samples were frozen in liquid nitrogen and ground with the 2000 Geno/Grinder®, and suspended in 200 μL ice-cold grinding buffer (50 mM Tris-Cl pH 7.5, 150 mM NaCl, 0.1% NP-40, 0.1% SDS, 25 mM β-ME, 1x plant protease inhibitor cocktail (Sigma-Aldrich®)).

Construct:

Article Title: Conserved residues in the wheat (Triticum aestivum) NAM-A1 NAC domain are required for protein binding and when mutated lead to delayed peduncle and flag leaf senescence
Article Snippet: Co-immunoprecipitation The wild-type alleles of NAM-A1 in the pGWB21 vector (N-terminal Myc tag) and NAM-B1 in the pGWB12 vector (N-terminal FLAG tag) [ ] were co-infiltrated into N. benthamiana , alongside P19 at a total OD of 0.6 and individual OD of 0.2 per construct. .. Protein was extracted in a standard buffer (100 mM Tris-HCl, pH 7.5; 150 mM NaCl; 0.1% (v/v) Triton x-100; 1% (w/v) PVPP; 5 mM EDTA; 10% glycerol; 2 mM DTT; 1% protease inhibitors (Sigma)).

Recombinant:

Article Title: Tomato SlSAP3, a member of the stress‐associated protein family, is a positive regulator of immunity against Pseudomonas syringae pv. tomato DC3000
Article Snippet: .. Briefly, reactions (30 μL) contained 5 μg ubiquitin (Boston Biochem, Cambridge, MA, USA), 110 ng E1 (Merck Millipore, Darmstadt, Germany), 100 ng human recombinant UbcH2 (Abcam, Cambridge, UK), and purified 4 μg GST‐SlSAP3 in buffer (20 mM MOPS, pH 7.2, 100 mM KCl, 5 mM MgCl2 , 5 mM ATP and 10 mM DTT) and were incubated at 30 °C for 3 h. Reactions were stopped by adding SDS sample buffer and analysed by SDS‐PAGE. followed by immunoblotting using anti‐ubiquitin antibody (CalBiochem, La Jolla, CA, USA). .. Chemiluminescence signal was detected with SuperSignal West Pico PLUS Chemiluminescent Substrate kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's recommendations.

Staining:

Article Title: Identification and Characterization of microRNAs during Retinoic Acid-Induced Regeneration of a Molluscan Central Nervous System
Article Snippet: For staining procedures, CNS tissue sections and cultured cells underwent the same procedures modified from Lu and Tsourkas [ ]. .. Following these washes, cells and tissues were incubated in blocking buffer (3% bovine serum, 4× SSC, 0.1% Tween-20) for 30 min, then horseradish peroxidase (HRP) (Sigma) for 30 min at room temperature.

Article Title: Aminosalicylic acid reduces ER stress and Schwann cell death induced by MPZ mutations
Article Snippet: .. The membranes were then stained with 0.1% Ponceau S solution (Sigma-Aldrich) to ensure equal loading of the samples, and non-specific binding was blocked with a blocking buffer (Casein blocking buffer 10X; Sigma-Aldrich) for 1 h at room temperature. .. The membranes were incubated overnight at 4°C with the following primary antibodies: Anti-Myc-tag (1:2,000; ab9106, Abcam), anti-binding immunoglobulin protein (BiP, #3177), anti-C/EBP-homologous protein (CHOP, #5554), anti-cleaved caspase-3 (Asp175, #9661), anti-phospho-AKT (ser473, #9271), anti-phospho-SAPK/JNK (Thr183/Tyr185, #9251) (all 1:1,000) (all from Cell Signaling Technology) and anti-β-actin (A2228, Sigma-Aldrich).

Article Title: Transcriptional response of Lactococcus lactis during bacterial emulsification
Article Snippet: At room temperature and protected from light the cells were stained for 30 min, with Syto 60 (Thermo Fisher Scientific, Oregon, Hillsboro, USA) by adding 1 μl of the staining solution (5 mM in DMSO) to 1 ml of cell suspension (OD600 = 1). .. Buffer (10 mM phosphate) was separately prepared by adding carboxyfluorescein (200 ml buffer + 300 μl of 100 mM carboxyfluorescein (Sigma-Aldrich) stock solution in water) and kept out of the light.

SDS Page:

Article Title: Insights Into Dolphins' Immunology: Immuno-Phenotypic Study on Mediterranean and Atlantic Stranded Cetaceans
Article Snippet: .. The samples were diluted 1:1 in 2x Laemmli sample buffer (Sigma–Aldrich, St. Louis, MO, USA), boiled for 5 min at 95°C and separated by 12% SDS–PAGE in a mini-gel apparatus (Hoefer SE 260, GE Healthcare, UK) under denaturing and reducing conditions in according to Laemmli protocol ( ). .. Homogenate of human tonsil was used as a positive control to test the binding with specific antibodies.

Article Title: Perturbations of the ZED1 pseudokinase activate plant immunity
Article Snippet: Powdered frozen plant tissue was then suspended in 100 μL of Grinding Buffer (40 mM Tris pH = 7.5, 150 mM NaCl, 1 mM EDTA, 5 mM DTT, 1% Triton X-100, 0.1% sodium dodecyl sulfate) supplemented with a 1:500 dilution of plant protease inhibitor cocktail P9599 (Sigma). .. 15 uL from each of these extracts was then resolved by electrophoresis through 10% polyacrylamide SDS-PAGE gels prior to protein transfer to nitrocellulose membranes.

Article Title: Ubiquitin-mediated proteasome degradation regulates optic fissure fusion
Article Snippet: A fourth combination repeated Siah1:MYC pCIG2 (2 µg)+Nlz2:FLAG pCIG2 (1 µg) along with a treatment using 10 mM of MG132 in the last 4 h. Protein extraction was performed by adding Laemmli sample buffer (Sigma-Aldrich) and boiling the samples at 95°C for 10 min. .. Equal volume for each transfection was load on 10% SDS-PAGE and run for 90 min at 120 V. Then, they were transfer to a PVDF membrane for 90 min at 400 mA.

Article Title: Tomato SlSAP3, a member of the stress‐associated protein family, is a positive regulator of immunity against Pseudomonas syringae pv. tomato DC3000
Article Snippet: .. Briefly, reactions (30 μL) contained 5 μg ubiquitin (Boston Biochem, Cambridge, MA, USA), 110 ng E1 (Merck Millipore, Darmstadt, Germany), 100 ng human recombinant UbcH2 (Abcam, Cambridge, UK), and purified 4 μg GST‐SlSAP3 in buffer (20 mM MOPS, pH 7.2, 100 mM KCl, 5 mM MgCl2 , 5 mM ATP and 10 mM DTT) and were incubated at 30 °C for 3 h. Reactions were stopped by adding SDS sample buffer and analysed by SDS‐PAGE. followed by immunoblotting using anti‐ubiquitin antibody (CalBiochem, La Jolla, CA, USA). .. Chemiluminescence signal was detected with SuperSignal West Pico PLUS Chemiluminescent Substrate kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's recommendations.

Plasmid Preparation:

Article Title: Tomato SlSAP3, a member of the stress‐associated protein family, is a positive regulator of immunity against Pseudomonas syringae pv. tomato DC3000
Article Snippet: The resulting plasmid was introduced into the E. coli strain Rosetta DE3 and expression of GST‐SlSAP3 fusion was induced by adding of 1 mM isopropyl‐D‐thiogalactoside (IPTG) at 20 °C overnight. .. Briefly, reactions (30 μL) contained 5 μg ubiquitin (Boston Biochem, Cambridge, MA, USA), 110 ng E1 (Merck Millipore, Darmstadt, Germany), 100 ng human recombinant UbcH2 (Abcam, Cambridge, UK), and purified 4 μg GST‐SlSAP3 in buffer (20 mM MOPS, pH 7.2, 100 mM KCl, 5 mM MgCl2 , 5 mM ATP and 10 mM DTT) and were incubated at 30 °C for 3 h. Reactions were stopped by adding SDS sample buffer and analysed by SDS‐PAGE. followed by immunoblotting using anti‐ubiquitin antibody (CalBiochem, La Jolla, CA, USA).

Article Title: Conserved residues in the wheat (Triticum aestivum) NAM-A1 NAC domain are required for protein binding and when mutated lead to delayed peduncle and flag leaf senescence
Article Snippet: Co-immunoprecipitation The wild-type alleles of NAM-A1 in the pGWB21 vector (N-terminal Myc tag) and NAM-B1 in the pGWB12 vector (N-terminal FLAG tag) [ ] were co-infiltrated into N. benthamiana , alongside P19 at a total OD of 0.6 and individual OD of 0.2 per construct. .. Protein was extracted in a standard buffer (100 mM Tris-HCl, pH 7.5; 150 mM NaCl; 0.1% (v/v) Triton x-100; 1% (w/v) PVPP; 5 mM EDTA; 10% glycerol; 2 mM DTT; 1% protease inhibitors (Sigma)).

RNA Expression:

Article Title: Proteomics dataset containing proteins that obscure identification of TOPLESS interactors in Arabidopsis
Article Snippet: The two T1 plants with the highest RNA expression were selected by visually comparing the intensity of target to control bands ( A). .. Tissue samples were frozen in liquid nitrogen and ground with the 2000 Geno/Grinder®, and suspended in 200 μL ice-cold grinding buffer (50 mM Tris-Cl pH 7.5, 150 mM NaCl, 0.1% NP-40, 0.1% SDS, 25 mM β-ME, 1x plant protease inhibitor cocktail (Sigma-Aldrich®)).

Confocal Laser Scanning Microscopy:

Article Title: Transcriptional response of Lactococcus lactis during bacterial emulsification
Article Snippet: Paragraph title: Confocal Laser Scanning Microscopy (CLSM) ... Buffer (10 mM phosphate) was separately prepared by adding carboxyfluorescein (200 ml buffer + 300 μl of 100 mM carboxyfluorescein (Sigma-Aldrich) stock solution in water) and kept out of the light.

FLAG-tag:

Article Title: Conserved residues in the wheat (Triticum aestivum) NAM-A1 NAC domain are required for protein binding and when mutated lead to delayed peduncle and flag leaf senescence
Article Snippet: Co-immunoprecipitation The wild-type alleles of NAM-A1 in the pGWB21 vector (N-terminal Myc tag) and NAM-B1 in the pGWB12 vector (N-terminal FLAG tag) [ ] were co-infiltrated into N. benthamiana , alongside P19 at a total OD of 0.6 and individual OD of 0.2 per construct. .. Protein was extracted in a standard buffer (100 mM Tris-HCl, pH 7.5; 150 mM NaCl; 0.1% (v/v) Triton x-100; 1% (w/v) PVPP; 5 mM EDTA; 10% glycerol; 2 mM DTT; 1% protease inhibitors (Sigma)).

Lysis:

Article Title: Aminosalicylic acid reduces ER stress and Schwann cell death induced by MPZ mutations
Article Snippet: Total cells were harvested and lysed with RIPA lysis buffer (Thermo Fisher Scientific). .. The membranes were then stained with 0.1% Ponceau S solution (Sigma-Aldrich) to ensure equal loading of the samples, and non-specific binding was blocked with a blocking buffer (Casein blocking buffer 10X; Sigma-Aldrich) for 1 h at room temperature.

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  • 95
    Millipore hat assay buffer
    Inhibitory effects of TA on <t>HAT</t> activity. (A) A colorimetric assay was performed to measure HAT activity in the presence of TA at the indicated concentrations. The results are presented as percentages relative to the control sample, which was incubated without TA (left panel). The IC 50 value of TA inhibition of HAT activity was calculated using Prism software (right panel). (B) An in vitro HAT assay using autoradiography was performed to evaluate the effect of the anti-HAT activity of TA on the acetylation of a synthetic H4 peptide by a <t>HeLa</t> NE. The arrow indicates histone H4 (left panel). The autoradiography was quantified using Image J software (right panel). The values presented are the means ± SD of two independent experiments. Means with different superscript letters are significantly different, p
    Hat Assay Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hat assay buffer/product/Millipore
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    99
    Millipore sds loading buffer
    RanBP3L inhibits BMP-induced transcriptional responses. (A) Schematic diagram of RanBP3L and RanBP3. RanBP3L contains a Ran-binding domain (RanBD) with 71% identity to that of RanBP3. (B) RanBP3L binds with RanGTP. HEK293T cells were cotransfected with Myc-RanBP3L and FLAG-RanQ69L. RanBP3L was immunoprecipitated (IP) with anti-Myc antibody and then subjected to <t>SDS-PAGE</t> and Western blotting to detect RanBP3L-bound RanQ69L. WCL, whole-cell lysate. (C to E) RanBP3L inhibits BMP2-induced Id1-luc reporter activity. C3H10T1/2 (C), C2C12 (D), and ATDC5 cells (E) were transfected with RanBP3L, PPM1A, or RanBP3 together with Id1-luc reporter plasmids. BMP2 treatment and luciferase assays were done as described in Materials and Methods. RLU, relative light units. (F) RanBP3L does not affect TGF-β-induced SBE-luc reporter activity in HaCaT cells. HaCaT cells were transfected with RanBP3 or RanBP3L together with the SBE-luc reporter plasmid and then treated with 2 ng/ml TGF-β for 12 h. The experiment was carried out as described above for panels C to E. (G and H) Stable expression of hRanBP3L in C2C12 cells decreases mRNA levels of Id1 (G) and Id2 (H). Cells were treated with LDN193189 (BMP type I receptor inhibitor) (LDN) or BMP2 (50 ng/ml) for 6 h, and total RNA was extracted for qRT-PCR analysis. (I) RanBP3L knockdown efficacy was measured in C2C12 cells. C2C12 cells were transfected with RanBP3L siRNAs or control siRNA (siCTR), and total RNA was extracted for qRT-PCR analysis. (J) Knockdown of RanBP3L enhances BMP2-induced Id1-luc reporter activity. C2C12 cells were transfected with RanBP3L siRNAs or control siRNA, together with the Id1-luc reporter plasmid. (K and L) Knockdown of RanBP3L increases Id1 mRNA expression (K) and Id2 mRNA expression (L). C2C12 cells were transfected with the indicated siRNAs and treated with BMP2 (50 ng/ml) for 6 h or not treated.
    Sds Loading Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 172 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Millipore co immunoprecipitation co ip buffer
    Effect of HSD10-modification on CypD and how it may influence cancer cell growth and death. A. EV and HSD10 ov whole cell lysates were analyzed for CypD protein expression using immunoblotting. β-actin was used as the loading control, and CypD was normalized to actin (n = 4). B. Control shRNA and HSD10 shRNA whole cell lysates were analyzed for CypD protein expression using immunoblotting. β-actin was used as the loading control, and CypD was normalized to actin (n = 4). C. EV and HSD10 ov whole cell lysates were analyzed for HSD10-CypD complexes using <t>co-immunoprecipitation.</t> β-actin was used as the loading control for the input. The immunoblots demonstrate an increased HSD10-CypD interaction in PC-12 cells overexpressing HSD10 compared to EV cells. D. Confocal microscopy demonstrating immunofluorescence staining of HSD10 alone (red), CypD alone (green), and these two antigens co-localized (yellow) in EV and HSD10 ov cells. E. Immunofluorescence staining of HSD10 alone (red), mitochondrial marker SODII alone (green), and these two antigens co-localized (yellow) in HSD10 ov cells. F. Immunofluorescence staining of CypD alone (green), mitochondrial marker Hsp60 alone (red), and these two antigens co-localized (yellow) in HSD10 ov cells. Scale bar in F : 20 μm. G-H. Quantification of HSD10 and CypD fluorescence densities (depicted in D ) displayed as fold increase (n = 4). Data presented as mean ± SE. *P
    Co Immunoprecipitation Co Ip Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Millipore buffer c human smp30 gnl
    Overall structure of mouse <t>SMP30/GNL.</t> The structure is shown as a rainbow colored cartoon with N-terminus = blue and C-terminus = red. The divalent metal ion (labeled as M 2+ ) located at the center of the structure is shown as an orange sphere.
    Buffer C Human Smp30 Gnl, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Inhibitory effects of TA on HAT activity. (A) A colorimetric assay was performed to measure HAT activity in the presence of TA at the indicated concentrations. The results are presented as percentages relative to the control sample, which was incubated without TA (left panel). The IC 50 value of TA inhibition of HAT activity was calculated using Prism software (right panel). (B) An in vitro HAT assay using autoradiography was performed to evaluate the effect of the anti-HAT activity of TA on the acetylation of a synthetic H4 peptide by a HeLa NE. The arrow indicates histone H4 (left panel). The autoradiography was quantified using Image J software (right panel). The values presented are the means ± SD of two independent experiments. Means with different superscript letters are significantly different, p

    Journal: Molecular Metabolism

    Article Title: Tannic acid, a novel histone acetyltransferase inhibitor, prevents non-alcoholic fatty liver disease both in vivo and in vitro model

    doi: 10.1016/j.molmet.2018.11.001

    Figure Lengend Snippet: Inhibitory effects of TA on HAT activity. (A) A colorimetric assay was performed to measure HAT activity in the presence of TA at the indicated concentrations. The results are presented as percentages relative to the control sample, which was incubated without TA (left panel). The IC 50 value of TA inhibition of HAT activity was calculated using Prism software (right panel). (B) An in vitro HAT assay using autoradiography was performed to evaluate the effect of the anti-HAT activity of TA on the acetylation of a synthetic H4 peptide by a HeLa NE. The arrow indicates histone H4 (left panel). The autoradiography was quantified using Image J software (right panel). The values presented are the means ± SD of two independent experiments. Means with different superscript letters are significantly different, p

    Article Snippet: For autoradiography-based in vitro HAT activity assays, HeLa cell nuclear extract was incubated with HAT assay buffer (50 mM HEPES, pH 8.0; 10% glycerol; 1 mM DTT; 1 mM phenylmethylsulfonyl fluoride; and 10 mM sodium butyrate), 1 μL [3 H] acetyl-CoA, and 5 μg of biotinylated-H4 peptide (Millipore, Billerica, MA, USA) along with TA at the indicated concentrations at 30 °C for 1 h. The reactions were stopped by adding 5 × sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) sample buffer and samples were separated on 15% SDS–PAGE gels, followed by autoradiographic analysis.

    Techniques: HAT Assay, Activity Assay, Colorimetric Assay, Incubation, Inhibition, Software, In Vitro, Autoradiography

    RanBP3L inhibits BMP-induced transcriptional responses. (A) Schematic diagram of RanBP3L and RanBP3. RanBP3L contains a Ran-binding domain (RanBD) with 71% identity to that of RanBP3. (B) RanBP3L binds with RanGTP. HEK293T cells were cotransfected with Myc-RanBP3L and FLAG-RanQ69L. RanBP3L was immunoprecipitated (IP) with anti-Myc antibody and then subjected to SDS-PAGE and Western blotting to detect RanBP3L-bound RanQ69L. WCL, whole-cell lysate. (C to E) RanBP3L inhibits BMP2-induced Id1-luc reporter activity. C3H10T1/2 (C), C2C12 (D), and ATDC5 cells (E) were transfected with RanBP3L, PPM1A, or RanBP3 together with Id1-luc reporter plasmids. BMP2 treatment and luciferase assays were done as described in Materials and Methods. RLU, relative light units. (F) RanBP3L does not affect TGF-β-induced SBE-luc reporter activity in HaCaT cells. HaCaT cells were transfected with RanBP3 or RanBP3L together with the SBE-luc reporter plasmid and then treated with 2 ng/ml TGF-β for 12 h. The experiment was carried out as described above for panels C to E. (G and H) Stable expression of hRanBP3L in C2C12 cells decreases mRNA levels of Id1 (G) and Id2 (H). Cells were treated with LDN193189 (BMP type I receptor inhibitor) (LDN) or BMP2 (50 ng/ml) for 6 h, and total RNA was extracted for qRT-PCR analysis. (I) RanBP3L knockdown efficacy was measured in C2C12 cells. C2C12 cells were transfected with RanBP3L siRNAs or control siRNA (siCTR), and total RNA was extracted for qRT-PCR analysis. (J) Knockdown of RanBP3L enhances BMP2-induced Id1-luc reporter activity. C2C12 cells were transfected with RanBP3L siRNAs or control siRNA, together with the Id1-luc reporter plasmid. (K and L) Knockdown of RanBP3L increases Id1 mRNA expression (K) and Id2 mRNA expression (L). C2C12 cells were transfected with the indicated siRNAs and treated with BMP2 (50 ng/ml) for 6 h or not treated.

    Journal: Molecular and Cellular Biology

    Article Title: Nuclear Export of Smads by RanBP3L Regulates Bone Morphogenetic Protein Signaling and Mesenchymal Stem Cell Differentiation

    doi: 10.1128/MCB.00121-15

    Figure Lengend Snippet: RanBP3L inhibits BMP-induced transcriptional responses. (A) Schematic diagram of RanBP3L and RanBP3. RanBP3L contains a Ran-binding domain (RanBD) with 71% identity to that of RanBP3. (B) RanBP3L binds with RanGTP. HEK293T cells were cotransfected with Myc-RanBP3L and FLAG-RanQ69L. RanBP3L was immunoprecipitated (IP) with anti-Myc antibody and then subjected to SDS-PAGE and Western blotting to detect RanBP3L-bound RanQ69L. WCL, whole-cell lysate. (C to E) RanBP3L inhibits BMP2-induced Id1-luc reporter activity. C3H10T1/2 (C), C2C12 (D), and ATDC5 cells (E) were transfected with RanBP3L, PPM1A, or RanBP3 together with Id1-luc reporter plasmids. BMP2 treatment and luciferase assays were done as described in Materials and Methods. RLU, relative light units. (F) RanBP3L does not affect TGF-β-induced SBE-luc reporter activity in HaCaT cells. HaCaT cells were transfected with RanBP3 or RanBP3L together with the SBE-luc reporter plasmid and then treated with 2 ng/ml TGF-β for 12 h. The experiment was carried out as described above for panels C to E. (G and H) Stable expression of hRanBP3L in C2C12 cells decreases mRNA levels of Id1 (G) and Id2 (H). Cells were treated with LDN193189 (BMP type I receptor inhibitor) (LDN) or BMP2 (50 ng/ml) for 6 h, and total RNA was extracted for qRT-PCR analysis. (I) RanBP3L knockdown efficacy was measured in C2C12 cells. C2C12 cells were transfected with RanBP3L siRNAs or control siRNA (siCTR), and total RNA was extracted for qRT-PCR analysis. (J) Knockdown of RanBP3L enhances BMP2-induced Id1-luc reporter activity. C2C12 cells were transfected with RanBP3L siRNAs or control siRNA, together with the Id1-luc reporter plasmid. (K and L) Knockdown of RanBP3L increases Id1 mRNA expression (K) and Id2 mRNA expression (L). C2C12 cells were transfected with the indicated siRNAs and treated with BMP2 (50 ng/ml) for 6 h or not treated.

    Article Snippet: After several washes, precipitated proteins were eluted in SDS loading buffer, separated by SDS-PAGE, transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore), and detected by Western blotting with appropriate antibodies.

    Techniques: Binding Assay, Immunoprecipitation, SDS Page, Western Blot, Activity Assay, Transfection, Luciferase, Plasmid Preparation, Expressing, Quantitative RT-PCR

    Effect of HSD10-modification on CypD and how it may influence cancer cell growth and death. A. EV and HSD10 ov whole cell lysates were analyzed for CypD protein expression using immunoblotting. β-actin was used as the loading control, and CypD was normalized to actin (n = 4). B. Control shRNA and HSD10 shRNA whole cell lysates were analyzed for CypD protein expression using immunoblotting. β-actin was used as the loading control, and CypD was normalized to actin (n = 4). C. EV and HSD10 ov whole cell lysates were analyzed for HSD10-CypD complexes using co-immunoprecipitation. β-actin was used as the loading control for the input. The immunoblots demonstrate an increased HSD10-CypD interaction in PC-12 cells overexpressing HSD10 compared to EV cells. D. Confocal microscopy demonstrating immunofluorescence staining of HSD10 alone (red), CypD alone (green), and these two antigens co-localized (yellow) in EV and HSD10 ov cells. E. Immunofluorescence staining of HSD10 alone (red), mitochondrial marker SODII alone (green), and these two antigens co-localized (yellow) in HSD10 ov cells. F. Immunofluorescence staining of CypD alone (green), mitochondrial marker Hsp60 alone (red), and these two antigens co-localized (yellow) in HSD10 ov cells. Scale bar in F : 20 μm. G-H. Quantification of HSD10 and CypD fluorescence densities (depicted in D ) displayed as fold increase (n = 4). Data presented as mean ± SE. *P

    Journal: BMC Cancer

    Article Title: Overexpression of 17β-hydroxysteroid dehydrogenase type 10 increases pheochromocytoma cell growth and resistance to cell death

    doi: 10.1186/s12885-015-1173-5

    Figure Lengend Snippet: Effect of HSD10-modification on CypD and how it may influence cancer cell growth and death. A. EV and HSD10 ov whole cell lysates were analyzed for CypD protein expression using immunoblotting. β-actin was used as the loading control, and CypD was normalized to actin (n = 4). B. Control shRNA and HSD10 shRNA whole cell lysates were analyzed for CypD protein expression using immunoblotting. β-actin was used as the loading control, and CypD was normalized to actin (n = 4). C. EV and HSD10 ov whole cell lysates were analyzed for HSD10-CypD complexes using co-immunoprecipitation. β-actin was used as the loading control for the input. The immunoblots demonstrate an increased HSD10-CypD interaction in PC-12 cells overexpressing HSD10 compared to EV cells. D. Confocal microscopy demonstrating immunofluorescence staining of HSD10 alone (red), CypD alone (green), and these two antigens co-localized (yellow) in EV and HSD10 ov cells. E. Immunofluorescence staining of HSD10 alone (red), mitochondrial marker SODII alone (green), and these two antigens co-localized (yellow) in HSD10 ov cells. F. Immunofluorescence staining of CypD alone (green), mitochondrial marker Hsp60 alone (red), and these two antigens co-localized (yellow) in HSD10 ov cells. Scale bar in F : 20 μm. G-H. Quantification of HSD10 and CypD fluorescence densities (depicted in D ) displayed as fold increase (n = 4). Data presented as mean ± SE. *P

    Article Snippet: Cells were washed twice with pre-chilled PBS, and then harvested, centrifuged, and suspended in 250 μl Co-Immunoprecipitation (Co-IP) buffer containing 150 mM NaCl, 50 mM Tris–HCl, pH 7.4, 1 mM EDTA, 0.5% NP-40, and 100X protease inhibitor (EMD Millipore).

    Techniques: Modification, Expressing, shRNA, Immunoprecipitation, Western Blot, Confocal Microscopy, Immunofluorescence, Staining, Marker, Fluorescence

    Overall structure of mouse SMP30/GNL. The structure is shown as a rainbow colored cartoon with N-terminus = blue and C-terminus = red. The divalent metal ion (labeled as M 2+ ) located at the center of the structure is shown as an orange sphere.

    Journal: PLoS ONE

    Article Title: Structural Basis of the ?-Lactone-Ring Formation in Ascorbic Acid Biosynthesis by the Senescence Marker Protein-30/Gluconolactonase

    doi: 10.1371/journal.pone.0053706

    Figure Lengend Snippet: Overall structure of mouse SMP30/GNL. The structure is shown as a rainbow colored cartoon with N-terminus = blue and C-terminus = red. The divalent metal ion (labeled as M 2+ ) located at the center of the structure is shown as an orange sphere.

    Article Snippet: The obtained sample was applied to a Phenyl-Sepharose CL-4B column (SIGMA-ALDRICH, 50 ml) pre-equilibrated with buffer C. Human SMP30/GNL was eluted by a gradient from 0% to 100% of buffer D. The eluted fractions were concentrated using an Amicon Ultra-15 centrifugal filter unit (MWCO 10,000) (Millipore) and further purified using a HiLoad 16/60 Superdex 200 pg column (GE Healthcare, 120 ml) pre-equilibrated with buffer E. The eluted fractions were dialyzed against buffer D. Purified proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Coomassie brilliant blue (CBB) stain ( ).

    Techniques: Labeling

    Active site structures of mouse and human SMP30/GNL. ( A ) Mouse SMP30/GNL in the substrate-free form, ( B ) the mouse SMP30/GNL–1,5-AG complex, ( C ) the human SMP30/GNL–1,5-AG complex, ( D ) the mouse SMP30/GNL– d -glucose complex, and ( E ) the mouse SMP30/GNL–xylitol complex. Lid loop residues of mouse SMP30/GNL and human SMP30/GNL are shown in purple and blue, respectively. Carbon atoms of ligand residues for the divalent metal ion (orange sphere) and those for substrate/product analogues are shown in green and yellow, respectively. Other carbon atoms are shown in white.

    Journal: PLoS ONE

    Article Title: Structural Basis of the ?-Lactone-Ring Formation in Ascorbic Acid Biosynthesis by the Senescence Marker Protein-30/Gluconolactonase

    doi: 10.1371/journal.pone.0053706

    Figure Lengend Snippet: Active site structures of mouse and human SMP30/GNL. ( A ) Mouse SMP30/GNL in the substrate-free form, ( B ) the mouse SMP30/GNL–1,5-AG complex, ( C ) the human SMP30/GNL–1,5-AG complex, ( D ) the mouse SMP30/GNL– d -glucose complex, and ( E ) the mouse SMP30/GNL–xylitol complex. Lid loop residues of mouse SMP30/GNL and human SMP30/GNL are shown in purple and blue, respectively. Carbon atoms of ligand residues for the divalent metal ion (orange sphere) and those for substrate/product analogues are shown in green and yellow, respectively. Other carbon atoms are shown in white.

    Article Snippet: The obtained sample was applied to a Phenyl-Sepharose CL-4B column (SIGMA-ALDRICH, 50 ml) pre-equilibrated with buffer C. Human SMP30/GNL was eluted by a gradient from 0% to 100% of buffer D. The eluted fractions were concentrated using an Amicon Ultra-15 centrifugal filter unit (MWCO 10,000) (Millipore) and further purified using a HiLoad 16/60 Superdex 200 pg column (GE Healthcare, 120 ml) pre-equilibrated with buffer E. The eluted fractions were dialyzed against buffer D. Purified proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Coomassie brilliant blue (CBB) stain ( ).

    Techniques:

    Catalytic reaction of mouse SMP30/GNL. ( A ) The γ-lactone-forming reaction catalyzed by mouse SMP30/GNL. The product of the catalytic reaction of SMP30/GNL is l -gulono-γ-lactone, which is in turn converted to ascorbic acid by gluconolactone oxidase. ( B–D ) Substrate and product analogues used in this study: ( B ) xylitol, ( C ) 1,5-anhydro- d -glucitol (1,5-AG), and ( D ) d -glucose. Corresponding atoms in these molecules are marked in light orange and light yellow.

    Journal: PLoS ONE

    Article Title: Structural Basis of the ?-Lactone-Ring Formation in Ascorbic Acid Biosynthesis by the Senescence Marker Protein-30/Gluconolactonase

    doi: 10.1371/journal.pone.0053706

    Figure Lengend Snippet: Catalytic reaction of mouse SMP30/GNL. ( A ) The γ-lactone-forming reaction catalyzed by mouse SMP30/GNL. The product of the catalytic reaction of SMP30/GNL is l -gulono-γ-lactone, which is in turn converted to ascorbic acid by gluconolactone oxidase. ( B–D ) Substrate and product analogues used in this study: ( B ) xylitol, ( C ) 1,5-anhydro- d -glucitol (1,5-AG), and ( D ) d -glucose. Corresponding atoms in these molecules are marked in light orange and light yellow.

    Article Snippet: The obtained sample was applied to a Phenyl-Sepharose CL-4B column (SIGMA-ALDRICH, 50 ml) pre-equilibrated with buffer C. Human SMP30/GNL was eluted by a gradient from 0% to 100% of buffer D. The eluted fractions were concentrated using an Amicon Ultra-15 centrifugal filter unit (MWCO 10,000) (Millipore) and further purified using a HiLoad 16/60 Superdex 200 pg column (GE Healthcare, 120 ml) pre-equilibrated with buffer E. The eluted fractions were dialyzed against buffer D. Purified proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Coomassie brilliant blue (CBB) stain ( ).

    Techniques:

    Top view of the substrate-binding cavity. Surface representation of ( A ) mouse SMP30/GNL, ( B ) DFPase, ( C ) Drp35, and ( D ) PON. Residues in the lid loop of mouse SMP30/GNL and the divalent metal ions (labeled as M 2+ ) are shown in purple and orange, respectively. Structures of DFPase, Drp35, and PON are superposed onto mouse SMP30/GNL by the SSM fitting using the program Superpose [27] in the CCP4 program suite [20] , and all molecules are viewed from the same direction.

    Journal: PLoS ONE

    Article Title: Structural Basis of the ?-Lactone-Ring Formation in Ascorbic Acid Biosynthesis by the Senescence Marker Protein-30/Gluconolactonase

    doi: 10.1371/journal.pone.0053706

    Figure Lengend Snippet: Top view of the substrate-binding cavity. Surface representation of ( A ) mouse SMP30/GNL, ( B ) DFPase, ( C ) Drp35, and ( D ) PON. Residues in the lid loop of mouse SMP30/GNL and the divalent metal ions (labeled as M 2+ ) are shown in purple and orange, respectively. Structures of DFPase, Drp35, and PON are superposed onto mouse SMP30/GNL by the SSM fitting using the program Superpose [27] in the CCP4 program suite [20] , and all molecules are viewed from the same direction.

    Article Snippet: The obtained sample was applied to a Phenyl-Sepharose CL-4B column (SIGMA-ALDRICH, 50 ml) pre-equilibrated with buffer C. Human SMP30/GNL was eluted by a gradient from 0% to 100% of buffer D. The eluted fractions were concentrated using an Amicon Ultra-15 centrifugal filter unit (MWCO 10,000) (Millipore) and further purified using a HiLoad 16/60 Superdex 200 pg column (GE Healthcare, 120 ml) pre-equilibrated with buffer E. The eluted fractions were dialyzed against buffer D. Purified proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Coomassie brilliant blue (CBB) stain ( ).

    Techniques: Binding Assay, Labeling

    Proposed catalytic reaction mechanism of mouse SMP30/GNL. The divalent metal ion in the active site is labeled as M 2+ .

    Journal: PLoS ONE

    Article Title: Structural Basis of the ?-Lactone-Ring Formation in Ascorbic Acid Biosynthesis by the Senescence Marker Protein-30/Gluconolactonase

    doi: 10.1371/journal.pone.0053706

    Figure Lengend Snippet: Proposed catalytic reaction mechanism of mouse SMP30/GNL. The divalent metal ion in the active site is labeled as M 2+ .

    Article Snippet: The obtained sample was applied to a Phenyl-Sepharose CL-4B column (SIGMA-ALDRICH, 50 ml) pre-equilibrated with buffer C. Human SMP30/GNL was eluted by a gradient from 0% to 100% of buffer D. The eluted fractions were concentrated using an Amicon Ultra-15 centrifugal filter unit (MWCO 10,000) (Millipore) and further purified using a HiLoad 16/60 Superdex 200 pg column (GE Healthcare, 120 ml) pre-equilibrated with buffer E. The eluted fractions were dialyzed against buffer D. Purified proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Coomassie brilliant blue (CBB) stain ( ).

    Techniques: Labeling

    Structural comparison of the lid loops of mouse and human SMP30/GNL. ( A ) Lid loops of mouse and human SMP30/GNL in the substrate free form are shown in purple and blue, respectively. The divalent metal ion (labeled as M 2+ ) is shown in orange. ( B, C ) SA-omit maps (mFo-DFc maps) for the lid loop residues in mouse ( B ) and human ( C ) SMP30/GNL. The contour levels of the SA-omit maps are 3.0 σ and 2.0 σ for panels B and C, respectively. ( D ) Surface representation of mouse SMP30/GNL around the lid loop. The entrance for the substrate-binding cavity is indicated by an arrow. Residues in the lid loop are shown in purple.

    Journal: PLoS ONE

    Article Title: Structural Basis of the ?-Lactone-Ring Formation in Ascorbic Acid Biosynthesis by the Senescence Marker Protein-30/Gluconolactonase

    doi: 10.1371/journal.pone.0053706

    Figure Lengend Snippet: Structural comparison of the lid loops of mouse and human SMP30/GNL. ( A ) Lid loops of mouse and human SMP30/GNL in the substrate free form are shown in purple and blue, respectively. The divalent metal ion (labeled as M 2+ ) is shown in orange. ( B, C ) SA-omit maps (mFo-DFc maps) for the lid loop residues in mouse ( B ) and human ( C ) SMP30/GNL. The contour levels of the SA-omit maps are 3.0 σ and 2.0 σ for panels B and C, respectively. ( D ) Surface representation of mouse SMP30/GNL around the lid loop. The entrance for the substrate-binding cavity is indicated by an arrow. Residues in the lid loop are shown in purple.

    Article Snippet: The obtained sample was applied to a Phenyl-Sepharose CL-4B column (SIGMA-ALDRICH, 50 ml) pre-equilibrated with buffer C. Human SMP30/GNL was eluted by a gradient from 0% to 100% of buffer D. The eluted fractions were concentrated using an Amicon Ultra-15 centrifugal filter unit (MWCO 10,000) (Millipore) and further purified using a HiLoad 16/60 Superdex 200 pg column (GE Healthcare, 120 ml) pre-equilibrated with buffer E. The eluted fractions were dialyzed against buffer D. Purified proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Coomassie brilliant blue (CBB) stain ( ).

    Techniques: Labeling, Binding Assay