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Result of the recombinant protein yield and salidroside production in vitro . (a) SDS-PAGE analysis of the recombinant protein expression and purification. Lane M: protein molecular weight markers. Lane 1: total protein in uninduced strain. Lane 2: total protein in induced strain. Lane 3: concentrated protein after being purified by Ni affinity chromatography and <t>PD-10</t> column. Sample of 15 μ L was used for SDS-PAGE analysis. Target protein is indicated by arrows. (b) Result of salidroside production catalysed by UGT72B14 in vitro . Protein yield data of the optimized UGT72B14 was obtained by purified protein after induction of 7 h initiated with OD 600 = 0.325 ± 0.021 while that of the wild-type UGT72B14 was obtained by purified protein after induction of 9 h initiated with OD 600 = 0.632 ± 0.031. The recombinant enzyme catalysis reaction system (100 μ L) contained 50 mM Tris-HCl (pH 7.5), 2 mM UDP-glucose, 250 μ M tyrosol, and the enzyme protein of 0.2 mg, proceeded for 30 min at 30°C, and terminated with 200 μ L MeOH. Data were presented as mean ± SD.
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Article Title: Expression of Codon-Optimized Plant Glycosyltransferase UGT72B14 in Escherichia coli Enhances Salidroside Production

Journal: BioMed Research International

doi: 10.1155/2016/9845927

Result of the recombinant protein yield and salidroside production in vitro . (a) SDS-PAGE analysis of the recombinant protein expression and purification. Lane M: protein molecular weight markers. Lane 1: total protein in uninduced strain. Lane 2: total protein in induced strain. Lane 3: concentrated protein after being purified by Ni affinity chromatography and PD-10 column. Sample of 15 μ L was used for SDS-PAGE analysis. Target protein is indicated by arrows. (b) Result of salidroside production catalysed by UGT72B14 in vitro . Protein yield data of the optimized UGT72B14 was obtained by purified protein after induction of 7 h initiated with OD 600 = 0.325 ± 0.021 while that of the wild-type UGT72B14 was obtained by purified protein after induction of 9 h initiated with OD 600 = 0.632 ± 0.031. The recombinant enzyme catalysis reaction system (100 μ L) contained 50 mM Tris-HCl (pH 7.5), 2 mM UDP-glucose, 250 μ M tyrosol, and the enzyme protein of 0.2 mg, proceeded for 30 min at 30°C, and terminated with 200 μ L MeOH. Data were presented as mean ± SD.
Figure Legend Snippet: Result of the recombinant protein yield and salidroside production in vitro . (a) SDS-PAGE analysis of the recombinant protein expression and purification. Lane M: protein molecular weight markers. Lane 1: total protein in uninduced strain. Lane 2: total protein in induced strain. Lane 3: concentrated protein after being purified by Ni affinity chromatography and PD-10 column. Sample of 15 μ L was used for SDS-PAGE analysis. Target protein is indicated by arrows. (b) Result of salidroside production catalysed by UGT72B14 in vitro . Protein yield data of the optimized UGT72B14 was obtained by purified protein after induction of 7 h initiated with OD 600 = 0.325 ± 0.021 while that of the wild-type UGT72B14 was obtained by purified protein after induction of 9 h initiated with OD 600 = 0.632 ± 0.031. The recombinant enzyme catalysis reaction system (100 μ L) contained 50 mM Tris-HCl (pH 7.5), 2 mM UDP-glucose, 250 μ M tyrosol, and the enzyme protein of 0.2 mg, proceeded for 30 min at 30°C, and terminated with 200 μ L MeOH. Data were presented as mean ± SD.

Techniques Used: Recombinant, In Vitro, SDS Page, Expressing, Purification, Molecular Weight, Affinity Chromatography

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Centrifugation:

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Filtration:

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Mouse Assay:

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Bradford Assay:

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Construct:

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Incubation:

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BIA-KA:

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Modification:

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Transformation Assay:

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High Performance Liquid Chromatography:

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Immunohistochemistry:

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Buffer Exchange:

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Cell Culture:

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Generated:

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other:

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Protein Concentration:

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Polymerase Chain Reaction:

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Sonication:

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Injection:

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Recombinant:

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Article Snippet: Paragraph title: Purification of recombinant AnaPX. ... The active fractions were pooled and desalted by a PD 10 column (Amersham Pharmacia Biotech, NJ).

Article Title: Heparan sulfate and syndecan-1 are essential in maintaining murine and human intestinal epithelial barrier function
Article Snippet: Briefly, FGF2 (0.5 mg, E. coli recombinant material; Selective Genetics Inc.) was protected with heparin (0.4 mg) in 0.2 M HEPES buffer (pH 8.4) and mixed with biotin hydrazide (long arm, water-soluble, 40 μg; Pierce Biotechnology) in a final volume of 0.2 ml. .. The sample was desalted on a PD-10 column (Amersham Pharmacia Biotech) equilibrated with 20 mM HEPES buffer (pH 7.4) containing 0.2% BSA.

Nucleic Acid Electrophoresis:

Article Title: Molecular Characterization of a Novel Peroxidase from the Cyanobacterium Anabaena sp. Strain PCC 7120
Article Snippet: The active fractions were pooled and desalted by a PD 10 column (Amersham Pharmacia Biotech, NJ). .. The active fractions containing purified enzyme were pooled and desalted by a PD 10 column.

In Vivo:

Article Title: Age-related Changes in the Hepatic Microcirculation in Mice
Article Snippet: Paragraph title: 2.2. In vivo microscopy ... Unreacted dye was removed by filtration through a PD-10 column (Sephadex G-25, Amersham Pharmacia Biotech, Uppsala, Sweden) equilibrated and eluted with PBS.

Fluorescence:

Article Title: A Single Mutation in the Non-Amyloidogenic Region of IAPP (Amylin) Greatly Reduces Toxicity
Article Snippet: Nonencapsulated carboxyfluorescein was removed from the vesicles through size exclusion chromatography using a PD-10 column (Amersham Pharmacia Biotech, Uppsala, Sweden). .. Nonencapsulated carboxyfluorescein was removed from the vesicles through size exclusion chromatography using a PD-10 column (Amersham Pharmacia Biotech, Uppsala, Sweden).

Mutagenesis:

Article Title: Identification of Complement Regulatory Domains in Vaccinia Virus Complement Control Protein
Article Snippet: The cells were centrifuged, resuspended in 400 ml of BMMY (BMGY containing 0.5% methanol, but without 1% glycerol), and incubated at 30°C for an additional 96 h with vigorous shaking; 0.5% methanol was added after every 24 h. After incubation, cells were pelleted and the supernatant containing the VCP mutant was collected for purification. .. For further purification, CCP 1-3 and CCP 1-2 mutants were passed through a PD-10 column (Amersham Pharmacia Biotech) in 10 mM sodium phosphate (pH 7.4), loaded onto a heparin-agarose column in the same buffer, and eluted with 500 mM NaCl.

Article Title: Identification of Complement Regulatory Domains in Vaccinia Virus Complement Control Protein
Article Snippet: CCP 3-4 mutant was purified by being loaded onto DEAE-Sephacel in 10 mM sodium phosphate, pH 7.4, containing 150 mM NaCl. .. The flowthrough containing CCP 3-4 mutant was collected and buffer exchange was performed using a PD-10 column preequilibrated with 5 mM sodium acetate, pH 4.0. .. The sample was then loaded onto a Mono S column, and the bound proteins were eluted with a linear salt gradient of 0 to 500 mM NaCl.

Isolation:

Article Title: APE1/Ref-1 as a Serological Biomarker for the Detection of Bladder Cancer
Article Snippet: Following induction with isopropyl β-D-1-thiogalactopyranoside (IPTG), the cells were sonicated in lysis buffer (100 mM NaCl, 20 mM HEPES), and the recombinant protein was purified on a nickel-nitrilotriacetic acid agarose column (Qiagen, Valencia, CA). .. After washing, the isolated APE1/Ref-1 protein was eluted with 250 mM imidazole buffer followed by desalting on a PD-10 column (Amersham Pharmacia Biotech, Liverpool, UK) in PBS, and frozen in 10% glycerol at −80°C. .. This protein was subsequently used for the standard curve in the APE1/Ref-1 ELISA.

Article Title: Tat-Mediated p66shc Transduction Decreased Phosphorylation of Endothelial Nitric Oxide Synthase in Endothelial Cells
Article Snippet: The human p66shc was isolated from Xpress-tagged p66shc cDNA in pcDNA3/1/His A (Invitrogen, Carlsbad, CA, USA) [ ] by PCR using the following two primers; the sense primer was 5'-CGG GAT CCC GGA ATT CGG CTT ATG GAT CTC C-3' (containing an EcoRI restriction site), and the antisense primer was 5'-CGA AGC TTT CAC AGT TTC CGC TCC ACA GG-3' (containing a HindIII restriction site). .. After washing, TAT-p66shc was eluted using 250 mM imidazole containing Buffer Z followed by desalting on a PD-10 column (Amersham Pharmacia Biotech, Piscataway, NJ, USA) in PBS.

Article Title: Characterization of Synthetic Hydroxyproline-Rich Proteoglycans with Arabinogalactan Protein and Extensin Motifs in Arabidopsis
Article Snippet: Paragraph title: Isolation and Biochemical Analysis of the Oligopeptides ... Finally, the samples were desalted using a PD-10 column (Sephadex G-25 medium; Amersham-Pharmacia Biotech) and freeze dried.

Protein Quantitation:

Article Title: Metal stoichiometry and functional studies of the diphtheria toxin repressor
Article Snippet: Paragraph title: Protein Quantitation. ... The dry weight of DtxR was determined by desalting samples of DtxR (7.5 mg) into water by using a PD-10 column (Amersham–Pharmacia Biotech).

Size-exclusion Chromatography:

Article Title: Does cholesterol suppress the antimicrobial peptide induced disruption of lipid raft containing membranes?
Article Snippet: The mixture was then kept at a constant 60 °C (well above the chain melting temperature of both lipids used) and was passed twenty-one times through a stainless steel extruder containing two nylon filters and a polycarbonate membrane containing 100 nm pores obtained from Fisher Scientific (Wayne, Mi) to produce a homogenous mixture of large unilamellar vesicles (LUVs). .. Non-encapsulated carboxyfluorescein was removed from the vesicle solution through size exclusion chromatography using a PD-10 column (Amersham Pharmacia Biotech, Uppsala, Sweden). .. LUV solution subsequently contained 10 mM sodium phosphate buffer and 100 mM NaCl at pH 7.5.

Article Title: A Single Mutation in the Non-Amyloidogenic Region of IAPP (Amylin) Greatly Reduces Toxicity
Article Snippet: For the dye leakage experiments, carboxyfluorescein containing POPG vesicles were prepared by rehydrating the dried lipid film in 50 mM sodium phosphate buffer (pH 7.5) containing 40 mM carboxyfluorescein, adjusted to pH 7.5 by the addition of sodium hydroxide. .. Nonencapsulated carboxyfluorescein was removed from the vesicles through size exclusion chromatography using a PD-10 column (Amersham Pharmacia Biotech, Uppsala, Sweden). .. Vesicle solutions were used immediately; and a fresh vesicle solution was used for each experiment.

Article Title: Single nucleotide polymorphism detection by combinatorial fluorescence energy transfer tags and biotinylated dideoxynucleotides
Article Snippet: To synthesize the CFET tag containing Alexa 594, 10–12 nmol FAM-labeled oligonucleotides containing an amino linker in 40 µl of 0.25 M Na2 CO3 /NaHCO3 buffer pH 9.0, were incubated for 10 h at room temperature with an ∼27-fold excess of the Alexa 594 NHS ester in 12 µl of anhydrous dimethylsulfoxide. .. Unreacted dye was removed by size exclusion chromatography on a PD-10 column (Amersham Pharmacia Biotech, Piscataway, NJ). .. The CFET tags were then purified by gel electrophoresis and desalted with an oligonucleotide purification cartridge.

Labeling:

Article Title: Age-related Changes in the Hepatic Microcirculation in Mice
Article Snippet: Unreacted dye was removed by filtration through a PD-10 column (Sephadex G-25, Amersham Pharmacia Biotech, Uppsala, Sweden) equilibrated and eluted with PBS. .. To test the effect of AGE on subsequent endocytosis of another scavenger receptor ligand, a saturation dose of AGE-BSA (8.6 mg/ml, 0.1 ml) was injected through a mesenteric vein followed 10 min later by injection of TRITC-FSA (6 mg/ml, 0.1 ml) and the uptake of the latter monitored by in vivo microscopy.

Article Title: Site-specific dynamic nuclear polarization of hydration water as a generally applicable approach to monitor protein aggregation
Article Snippet: Paragraph title: Spin Labeling tau ... Excess spin label was removed by buffer exchange using a PD-10 column (Amersham Pharmacia Biotech) preequilibrated in storage buffer (20 mM sodium phosphate @ pH 7, 100 mM NaCl, 0.1 mM EDTA).

Article Title: Heparan sulfate and syndecan-1 are essential in maintaining murine and human intestinal epithelial barrier function
Article Snippet: The sample was desalted on a PD-10 column (Amersham Pharmacia Biotech) equilibrated with 20 mM HEPES buffer (pH 7.4) containing 0.2% BSA. .. The sample was desalted on a PD-10 column (Amersham Pharmacia Biotech) equilibrated with 20 mM HEPES buffer (pH 7.4) containing 0.2% BSA.

Purification:

Article Title: Expression of Codon-Optimized Plant Glycosyltransferase UGT72B14 in Escherichia coli Enhances Salidroside Production
Article Snippet: After washing with elution buffer (20 mM Tris, pH7.5, 300 mM NaCl, and 20 mM imidazole), unbound contaminant proteins were removed. .. His-tagged proteins were eluted with washing buffer (20 mM Tris, pH7.5, 300 mM NaCl, and 300 mM imidazole) and purified using a PD-10 column (Amersham Pharmacia Biotech, Uppsala, Sweden) and then concentrated by a centrifugal filter unit (YM-50 kD, Millipore). .. The efficiency of purification was monitored by SDS-PAGE analysis.

Article Title: APE1/Ref-1 as a Serological Biomarker for the Detection of Bladder Cancer
Article Snippet: Following induction with isopropyl β-D-1-thiogalactopyranoside (IPTG), the cells were sonicated in lysis buffer (100 mM NaCl, 20 mM HEPES), and the recombinant protein was purified on a nickel-nitrilotriacetic acid agarose column (Qiagen, Valencia, CA). .. After washing, the isolated APE1/Ref-1 protein was eluted with 250 mM imidazole buffer followed by desalting on a PD-10 column (Amersham Pharmacia Biotech, Liverpool, UK) in PBS, and frozen in 10% glycerol at −80°C.

Article Title: Identification of Complement Regulatory Domains in Vaccinia Virus Complement Control Protein
Article Snippet: The pellets were then suspended and dialyzed in PBS. .. For further purification, CCP 1-3 and CCP 1-2 mutants were passed through a PD-10 column (Amersham Pharmacia Biotech) in 10 mM sodium phosphate (pH 7.4), loaded onto a heparin-agarose column in the same buffer, and eluted with 500 mM NaCl. .. The eluted proteins were then exchanged into 20 mM Tris (pH 7.9), loaded onto a Mono Q column, and eluted with a linear gradient of 0 to 500 mM NaCl.

Article Title: Tat-Mediated p66shc Transduction Decreased Phosphorylation of Endothelial Nitric Oxide Synthase in Endothelial Cells
Article Snippet: Paragraph title: Recombinant protein expression and purification ... After washing, TAT-p66shc was eluted using 250 mM imidazole containing Buffer Z followed by desalting on a PD-10 column (Amersham Pharmacia Biotech, Piscataway, NJ, USA) in PBS.

Article Title: Apurinic/apyrimidinic endonuclease 1 inhibits protein kinase C-mediated p66shc phosphorylation and vasoconstriction
Article Snippet: Paragraph title: 2.5. Recombinant Tat-APE1 protein expression and purification ... After washing, Tat-APE1 was eluted using 250 mM imidazole-containing buffer Z followed by desalting on a PD-10 column (Amersham Pharmacia Biotech, Piscataway, NJ, USA) in PBS.

Article Title: Galectin-1 Induces Reversible Phosphatidylserine Exposure at the Plasma Membrane
Article Snippet: The expression and purification of human Gal-1 was described previously ( ). .. Gal-1 was alkylated with iodoacetamide followed by removal of free iodoacetamide using a PD-10 column (Amersham Pharmacia Biotech, Piscataway, NJ) equilibrated in PBS, as outlined previously ( ).

Article Title: Molecular Characterization of a Novel Peroxidase from the Cyanobacterium Anabaena sp. Strain PCC 7120
Article Snippet: Paragraph title: Purification of recombinant AnaPX. ... The active fractions were pooled and desalted by a PD 10 column (Amersham Pharmacia Biotech, NJ).

Article Title: Dual Roles of Helicobacter pylori NapA in Inducing and Combating Oxidative Stress
Article Snippet: Briefly, ferrous ammonium sulfate (Sigma) was added to a 100-μg/ml NapA preparation in 0.1 mM morpholinepropanesulfonic acid (MOPS)-NaOH buffer (pH 7.0) at a final concentration of 1 mM. .. The mixture was kept under a nitrogen atmosphere and incubated at room temperature for 1 h. The Fe-loaded NapA was then purified on a PD-10 column (Amersham Pharmacia Biotech) equilibrated with 20 mM MOPS-NaOH buffer (pH 7.0). .. The eluted protein was concentrated by an Centricon YM-50 (Millipore Corp.).

Protein Purification:

Article Title: Expression of Codon-Optimized Plant Glycosyltransferase UGT72B14 in Escherichia coli Enhances Salidroside Production
Article Snippet: Paragraph title: 2.4. Protein Purification and Measurement of Enzyme Activity ... His-tagged proteins were eluted with washing buffer (20 mM Tris, pH7.5, 300 mM NaCl, and 300 mM imidazole) and purified using a PD-10 column (Amersham Pharmacia Biotech, Uppsala, Sweden) and then concentrated by a centrifugal filter unit (YM-50 kD, Millipore).

Sequencing:

Article Title: Characterization of Synthetic Hydroxyproline-Rich Proteoglycans with Arabinogalactan Protein and Extensin Motifs in Arabidopsis
Article Snippet: Finally, the samples were desalted using a PD-10 column (Sephadex G-25 medium; Amersham-Pharmacia Biotech) and freeze dried. .. Finally, the samples were desalted using a PD-10 column (Sephadex G-25 medium; Amersham-Pharmacia Biotech) and freeze dried.

Microscopy:

Article Title: Age-related Changes in the Hepatic Microcirculation in Mice
Article Snippet: Paragraph title: 2.2. In vivo microscopy ... Unreacted dye was removed by filtration through a PD-10 column (Sephadex G-25, Amersham Pharmacia Biotech, Uppsala, Sweden) equilibrated and eluted with PBS.

Lysis:

Article Title: Expression of Codon-Optimized Plant Glycosyltransferase UGT72B14 in Escherichia coli Enhances Salidroside Production
Article Snippet: When the OD600 reached 0.3 ± 0.1, E. coli harbouring the optimized UGT72B14 plasmid was induced at 20°C with shaking at 120 rpm as described above for a further 6–8 h. Cell pellets were harvested by centrifugation (8,000 ×g; 10 min; 4°C) and resuspended in lysis buffer (20 mM Tris, pH7.5, 300 mM NaCl, 20 mM imidazole, 1 mg/mL leupeptin, 2 mg/mL aprotinin, and 100 mg/mL lysozyme). .. His-tagged proteins were eluted with washing buffer (20 mM Tris, pH7.5, 300 mM NaCl, and 300 mM imidazole) and purified using a PD-10 column (Amersham Pharmacia Biotech, Uppsala, Sweden) and then concentrated by a centrifugal filter unit (YM-50 kD, Millipore).

Article Title: APE1/Ref-1 as a Serological Biomarker for the Detection of Bladder Cancer
Article Snippet: Following induction with isopropyl β-D-1-thiogalactopyranoside (IPTG), the cells were sonicated in lysis buffer (100 mM NaCl, 20 mM HEPES), and the recombinant protein was purified on a nickel-nitrilotriacetic acid agarose column (Qiagen, Valencia, CA). .. After washing, the isolated APE1/Ref-1 protein was eluted with 250 mM imidazole buffer followed by desalting on a PD-10 column (Amersham Pharmacia Biotech, Liverpool, UK) in PBS, and frozen in 10% glycerol at −80°C.

Countercurrent Chromatography:

Article Title: Tat-Mediated p66shc Transduction Decreased Phosphorylation of Endothelial Nitric Oxide Synthase in Endothelial Cells
Article Snippet: The human p66shc was isolated from Xpress-tagged p66shc cDNA in pcDNA3/1/His A (Invitrogen, Carlsbad, CA, USA) [ ] by PCR using the following two primers; the sense primer was 5'-CGG GAT CCC GGA ATT CGG CTT ATG GAT CTC C-3' (containing an EcoRI restriction site), and the antisense primer was 5'-CGA AGC TTT CAC AGT TTC CGC TCC ACA GG-3' (containing a HindIII restriction site). .. After washing, TAT-p66shc was eluted using 250 mM imidazole containing Buffer Z followed by desalting on a PD-10 column (Amersham Pharmacia Biotech, Piscataway, NJ, USA) in PBS.

SDS Page:

Article Title: Molecular Characterization of a Novel Peroxidase from the Cyanobacterium Anabaena sp. Strain PCC 7120
Article Snippet: The active fractions were pooled and desalted by a PD 10 column (Amersham Pharmacia Biotech, NJ). .. The active fractions containing purified enzyme were pooled and desalted by a PD 10 column.

Article Title: Site-specific dynamic nuclear polarization of hydration water as a generally applicable approach to monitor protein aggregation
Article Snippet: Excess spin label was removed by buffer exchange using a PD-10 column (Amersham Pharmacia Biotech) preequilibrated in storage buffer (20 mM sodium phosphate @ pH 7, 100 mM NaCl, 0.1 mM EDTA). .. Excess spin label was removed by buffer exchange using a PD-10 column (Amersham Pharmacia Biotech) preequilibrated in storage buffer (20 mM sodium phosphate @ pH 7, 100 mM NaCl, 0.1 mM EDTA).

Plasmid Preparation:

Article Title: Expression of Codon-Optimized Plant Glycosyltransferase UGT72B14 in Escherichia coli Enhances Salidroside Production
Article Snippet: When the OD600 reached 0.3 ± 0.1, E. coli harbouring the optimized UGT72B14 plasmid was induced at 20°C with shaking at 120 rpm as described above for a further 6–8 h. Cell pellets were harvested by centrifugation (8,000 ×g; 10 min; 4°C) and resuspended in lysis buffer (20 mM Tris, pH7.5, 300 mM NaCl, 20 mM imidazole, 1 mg/mL leupeptin, 2 mg/mL aprotinin, and 100 mg/mL lysozyme). .. His-tagged proteins were eluted with washing buffer (20 mM Tris, pH7.5, 300 mM NaCl, and 300 mM imidazole) and purified using a PD-10 column (Amersham Pharmacia Biotech, Uppsala, Sweden) and then concentrated by a centrifugal filter unit (YM-50 kD, Millipore).

Article Title: APE1/Ref-1 as a Serological Biomarker for the Detection of Bladder Cancer
Article Snippet: Human full length APE1/Ref-1 DNA was inserted into the pET28b expression vector (Novagen, Gibbstown, NJ), containing a 6-histidine tag for easy purification [ ]. pET28b-APE1/Ref-1 plasmids were then transformed into the BL21(DE3) strain of Escherichia coli . .. After washing, the isolated APE1/Ref-1 protein was eluted with 250 mM imidazole buffer followed by desalting on a PD-10 column (Amersham Pharmacia Biotech, Liverpool, UK) in PBS, and frozen in 10% glycerol at −80°C.

Article Title: Development of 3D in vitro platform technology to engineer mesenchymal stem cells
Article Snippet: The plasmid DNA-BMP-2 and RITC were mixed in 0.2 M sodium carbonate-buffered solution (pH 9.7) at 4°C for 12 hours, both at concentrations of 1 mg/mL in order to prepare fluorescent-labeled plasmid DNA. .. The RITC-labeled DNA was separated from the residual RITC by gel filtration of a PD 10 column (Amersham Pharmacia Biotech, Tokyo, Japan), followed by ethanol precipitation to obtain the RITC-labeled plasmid DNA. .. According to the procedure mentioned above, the cationized dextran was mixed with the RITC-labeled plasmid DNA in PBS at the N/P ratio of 5 to prepare the DNA nanoparticles.

Article Title: Tat-Mediated p66shc Transduction Decreased Phosphorylation of Endothelial Nitric Oxide Synthase in Endothelial Cells
Article Snippet: After digesting with EcoRI and Hind III, the full length p66shc constructs were cloned into the pTAT bacterial expression vector (pTAT-2.1, kindly donated by Steven Dowdy), which contains a six-histidine tag, for easy purification. p66shc (S36A) cDNA was kindly donated by Kaikobad Irani (Pittsburgh University). pTAT-p66shc plasmids were then transformed into the BL21 (DE3) strain of E. coli . .. After washing, TAT-p66shc was eluted using 250 mM imidazole containing Buffer Z followed by desalting on a PD-10 column (Amersham Pharmacia Biotech, Piscataway, NJ, USA) in PBS.

Article Title: Apurinic/apyrimidinic endonuclease 1 inhibits protein kinase C-mediated p66shc phosphorylation and vasoconstriction
Article Snippet: Full-length human APE1 cDNA was cloned into the pTAT bacterial expression vector (pTAT-2.1, kindly provided by Steven Dowdy), which contains a six-histidine tag, for easy purification. pTAT-APE1 plasmids were then transformed into the BL21(DE3) strain of Escherichia coli . .. After washing, Tat-APE1 was eluted using 250 mM imidazole-containing buffer Z followed by desalting on a PD-10 column (Amersham Pharmacia Biotech, Piscataway, NJ, USA) in PBS.

Software:

Article Title: Heparan sulfate and syndecan-1 are essential in maintaining murine and human intestinal epithelial barrier function
Article Snippet: The sample was desalted on a PD-10 column (Amersham Pharmacia Biotech) equilibrated with 20 mM HEPES buffer (pH 7.4) containing 0.2% BSA. .. The sample was desalted on a PD-10 column (Amersham Pharmacia Biotech) equilibrated with 20 mM HEPES buffer (pH 7.4) containing 0.2% BSA.

Binding Assay:

Article Title: Characterization of Synthetic Hydroxyproline-Rich Proteoglycans with Arabinogalactan Protein and Extensin Motifs in Arabidopsis
Article Snippet: The samples were incubated for 2 to 3 h at room temperature and the column was washed with 20 volumes of the binding buffer. .. Finally, the samples were desalted using a PD-10 column (Sephadex G-25 medium; Amersham-Pharmacia Biotech) and freeze dried.

In Vitro:

Article Title: Development of 3D in vitro platform technology to engineer mesenchymal stem cells
Article Snippet: Paragraph title: In vitro release test of plasmid DNA ... The RITC-labeled DNA was separated from the residual RITC by gel filtration of a PD 10 column (Amersham Pharmacia Biotech, Tokyo, Japan), followed by ethanol precipitation to obtain the RITC-labeled plasmid DNA.

Ethanol Precipitation:

Article Title: Development of 3D in vitro platform technology to engineer mesenchymal stem cells
Article Snippet: The plasmid DNA-BMP-2 and RITC were mixed in 0.2 M sodium carbonate-buffered solution (pH 9.7) at 4°C for 12 hours, both at concentrations of 1 mg/mL in order to prepare fluorescent-labeled plasmid DNA. .. The RITC-labeled DNA was separated from the residual RITC by gel filtration of a PD 10 column (Amersham Pharmacia Biotech, Tokyo, Japan), followed by ethanol precipitation to obtain the RITC-labeled plasmid DNA. .. According to the procedure mentioned above, the cationized dextran was mixed with the RITC-labeled plasmid DNA in PBS at the N/P ratio of 5 to prepare the DNA nanoparticles.

Spectrophotometry:

Article Title: Effect of Nutrient Starvation on the Cellular Composition and Metabolic Capacity of Saccharomyces cerevisiae
Article Snippet: The crude extracts were used immediately for analysis of enzyme activity at 30°C either with a COBAS Fara Autoanalyser (Roche), with a Fluostar spectrophotometric plate reader (BMG Labtechnologies) equipped with an internal reagent pump for addition of the starting substrate, or manually with a Shimadzu spectrophotometer (UV-240 with the option program/interface OPI-1). .. The extracts used for determination of glycerol phosphate dehydrogenase and glycerol phosphate phosphatase activities were desalted on a PD-10 column (Amersham Pharmacia Biotech) prior to measurement.

Evaporation:

Article Title: Does cholesterol suppress the antimicrobial peptide induced disruption of lipid raft containing membranes?
Article Snippet: The appropriate volumes of stock solution for each sample were mixed in a small, round-bottomed flask and the solvent was removed by evaporation over a gentle stream of dry nitrogen gas. .. Non-encapsulated carboxyfluorescein was removed from the vesicle solution through size exclusion chromatography using a PD-10 column (Amersham Pharmacia Biotech, Uppsala, Sweden).

Concentration Assay:

Article Title: Expression of Codon-Optimized Plant Glycosyltransferase UGT72B14 in Escherichia coli Enhances Salidroside Production
Article Snippet: His-tagged proteins were eluted with washing buffer (20 mM Tris, pH7.5, 300 mM NaCl, and 300 mM imidazole) and purified using a PD-10 column (Amersham Pharmacia Biotech, Uppsala, Sweden) and then concentrated by a centrifugal filter unit (YM-50 kD, Millipore). .. The efficiency of purification was monitored by SDS-PAGE analysis.

Article Title: Does cholesterol suppress the antimicrobial peptide induced disruption of lipid raft containing membranes?
Article Snippet: After the complete removal of solvents, the dry lipid films were hydrated at room temperature in the same small, round-bottomed flask with carboxyfluorescein dye at a concentration of 70 mM in 10 mM pH 7.5 sodium phosphate buffer without NaCl. .. Non-encapsulated carboxyfluorescein was removed from the vesicle solution through size exclusion chromatography using a PD-10 column (Amersham Pharmacia Biotech, Uppsala, Sweden).

Article Title: Molecular Characterization of a Novel Peroxidase from the Cyanobacterium Anabaena sp. Strain PCC 7120
Article Snippet: The active fractions were pooled, solid ammonium sulfate was added to a 20% saturated concentration; fractions were subjected to a Toyopearl Butyl-650M column (2.5 by 20 cm; Tosoh Corp.) equilibrated with buffer A containing 20% saturated ammonium sulfate and eluted with a linear gradient of 20 to 0% saturated ammonium sulfate. .. The active fractions were pooled and desalted by a PD 10 column (Amersham Pharmacia Biotech, NJ).

Article Title: Metal stoichiometry and functional studies of the diphtheria toxin repressor
Article Snippet: Sample concentration was determined by the Bradford method before and after analysis; in all cases the concentrations before and after QAA were in good agreement. .. The dry weight of DtxR was determined by desalting samples of DtxR (7.5 mg) into water by using a PD-10 column (Amersham–Pharmacia Biotech).

Article Title: Dual Roles of Helicobacter pylori NapA in Inducing and Combating Oxidative Stress
Article Snippet: Briefly, ferrous ammonium sulfate (Sigma) was added to a 100-μg/ml NapA preparation in 0.1 mM morpholinepropanesulfonic acid (MOPS)-NaOH buffer (pH 7.0) at a final concentration of 1 mM. .. The mixture was kept under a nitrogen atmosphere and incubated at room temperature for 1 h. The Fe-loaded NapA was then purified on a PD-10 column (Amersham Pharmacia Biotech) equilibrated with 20 mM MOPS-NaOH buffer (pH 7.0).

Staining:

Article Title: Heparan sulfate and syndecan-1 are essential in maintaining murine and human intestinal epithelial barrier function
Article Snippet: Paragraph title: Immunohistochemistry: HS staining with biotinylated FGF. ... The sample was desalted on a PD-10 column (Amersham Pharmacia Biotech) equilibrated with 20 mM HEPES buffer (pH 7.4) containing 0.2% BSA.

Affinity Column:

Article Title: Characterization of Synthetic Hydroxyproline-Rich Proteoglycans with Arabinogalactan Protein and Extensin Motifs in Arabidopsis
Article Snippet: The EGFP positive fractions were solubilized in 1 mL of the binding buffer (phosphate-buffered saline 0.1 m , 0.15 m NaCl, pH 7.2) and applied into the affinity column. .. Finally, the samples were desalted using a PD-10 column (Sephadex G-25 medium; Amersham-Pharmacia Biotech) and freeze dried.

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    Amersham Pharmacia Biotech Ltd pd 10 column
    Result of the recombinant protein yield and salidroside production in vitro . (a) SDS-PAGE analysis of the recombinant protein expression and purification. Lane M: protein molecular weight markers. Lane 1: total protein in uninduced strain. Lane 2: total protein in induced strain. Lane 3: concentrated protein after being purified by Ni affinity chromatography and <t>PD-10</t> column. Sample of 15 μ L was used for SDS-PAGE analysis. Target protein is indicated by arrows. (b) Result of salidroside production catalysed by UGT72B14 in vitro . Protein yield data of the optimized UGT72B14 was obtained by purified protein after induction of 7 h initiated with OD 600 = 0.325 ± 0.021 while that of the wild-type UGT72B14 was obtained by purified protein after induction of 9 h initiated with OD 600 = 0.632 ± 0.031. The recombinant enzyme catalysis reaction system (100 μ L) contained 50 mM Tris-HCl (pH 7.5), 2 mM UDP-glucose, 250 μ M tyrosol, and the enzyme protein of 0.2 mg, proceeded for 30 min at 30°C, and terminated with 200 μ L MeOH. Data were presented as mean ± SD.
    Pd 10 Column, supplied by Amersham Pharmacia Biotech Ltd, used in various techniques. Bioz Stars score: 89/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pd 10 column/product/Amersham Pharmacia Biotech Ltd
    Average 89 stars, based on 13 article reviews
    Price from $9.99 to $1999.99
    pd 10 column - by Bioz Stars, 2020-01
    89/100 stars
      Buy from Supplier

    79
    Amersham Pharmacia Biotech Ltd pd 10 desalting workmate nickel sepharose columns
    Result of the recombinant protein yield and salidroside production in vitro . (a) SDS-PAGE analysis of the recombinant protein expression and purification. Lane M: protein molecular weight markers. Lane 1: total protein in uninduced strain. Lane 2: total protein in induced strain. Lane 3: concentrated protein after being purified by Ni affinity chromatography and <t>PD-10</t> column. Sample of 15 μ L was used for SDS-PAGE analysis. Target protein is indicated by arrows. (b) Result of salidroside production catalysed by UGT72B14 in vitro . Protein yield data of the optimized UGT72B14 was obtained by purified protein after induction of 7 h initiated with OD 600 = 0.325 ± 0.021 while that of the wild-type UGT72B14 was obtained by purified protein after induction of 9 h initiated with OD 600 = 0.632 ± 0.031. The recombinant enzyme catalysis reaction system (100 μ L) contained 50 mM Tris-HCl (pH 7.5), 2 mM UDP-glucose, 250 μ M tyrosol, and the enzyme protein of 0.2 mg, proceeded for 30 min at 30°C, and terminated with 200 μ L MeOH. Data were presented as mean ± SD.
    Pd 10 Desalting Workmate Nickel Sepharose Columns, supplied by Amersham Pharmacia Biotech Ltd, used in various techniques. Bioz Stars score: 79/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pd 10 desalting workmate nickel sepharose columns/product/Amersham Pharmacia Biotech Ltd
    Average 79 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pd 10 desalting workmate nickel sepharose columns - by Bioz Stars, 2020-01
    79/100 stars
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    79
    Amersham Pharmacia Biotech Ltd sephadex purified protein extracts
    Result of the recombinant protein yield and salidroside production in vitro . (a) SDS-PAGE analysis of the recombinant protein expression and purification. Lane M: protein molecular weight markers. Lane 1: total protein in uninduced strain. Lane 2: total protein in induced strain. Lane 3: concentrated protein after being purified by Ni affinity chromatography and <t>PD-10</t> column. Sample of 15 μ L was used for SDS-PAGE analysis. Target protein is indicated by arrows. (b) Result of salidroside production catalysed by UGT72B14 in vitro . Protein yield data of the optimized UGT72B14 was obtained by purified protein after induction of 7 h initiated with OD 600 = 0.325 ± 0.021 while that of the wild-type UGT72B14 was obtained by purified protein after induction of 9 h initiated with OD 600 = 0.632 ± 0.031. The recombinant enzyme catalysis reaction system (100 μ L) contained 50 mM Tris-HCl (pH 7.5), 2 mM UDP-glucose, 250 μ M tyrosol, and the enzyme protein of 0.2 mg, proceeded for 30 min at 30°C, and terminated with 200 μ L MeOH. Data were presented as mean ± SD.
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    Result of the recombinant protein yield and salidroside production in vitro . (a) SDS-PAGE analysis of the recombinant protein expression and purification. Lane M: protein molecular weight markers. Lane 1: total protein in uninduced strain. Lane 2: total protein in induced strain. Lane 3: concentrated protein after being purified by Ni affinity chromatography and PD-10 column. Sample of 15 μ L was used for SDS-PAGE analysis. Target protein is indicated by arrows. (b) Result of salidroside production catalysed by UGT72B14 in vitro . Protein yield data of the optimized UGT72B14 was obtained by purified protein after induction of 7 h initiated with OD 600 = 0.325 ± 0.021 while that of the wild-type UGT72B14 was obtained by purified protein after induction of 9 h initiated with OD 600 = 0.632 ± 0.031. The recombinant enzyme catalysis reaction system (100 μ L) contained 50 mM Tris-HCl (pH 7.5), 2 mM UDP-glucose, 250 μ M tyrosol, and the enzyme protein of 0.2 mg, proceeded for 30 min at 30°C, and terminated with 200 μ L MeOH. Data were presented as mean ± SD.

    Journal: BioMed Research International

    Article Title: Expression of Codon-Optimized Plant Glycosyltransferase UGT72B14 in Escherichia coli Enhances Salidroside Production

    doi: 10.1155/2016/9845927

    Figure Lengend Snippet: Result of the recombinant protein yield and salidroside production in vitro . (a) SDS-PAGE analysis of the recombinant protein expression and purification. Lane M: protein molecular weight markers. Lane 1: total protein in uninduced strain. Lane 2: total protein in induced strain. Lane 3: concentrated protein after being purified by Ni affinity chromatography and PD-10 column. Sample of 15 μ L was used for SDS-PAGE analysis. Target protein is indicated by arrows. (b) Result of salidroside production catalysed by UGT72B14 in vitro . Protein yield data of the optimized UGT72B14 was obtained by purified protein after induction of 7 h initiated with OD 600 = 0.325 ± 0.021 while that of the wild-type UGT72B14 was obtained by purified protein after induction of 9 h initiated with OD 600 = 0.632 ± 0.031. The recombinant enzyme catalysis reaction system (100 μ L) contained 50 mM Tris-HCl (pH 7.5), 2 mM UDP-glucose, 250 μ M tyrosol, and the enzyme protein of 0.2 mg, proceeded for 30 min at 30°C, and terminated with 200 μ L MeOH. Data were presented as mean ± SD.

    Article Snippet: His-tagged proteins were eluted with washing buffer (20 mM Tris, pH7.5, 300 mM NaCl, and 300 mM imidazole) and purified using a PD-10 column (Amersham Pharmacia Biotech, Uppsala, Sweden) and then concentrated by a centrifugal filter unit (YM-50 kD, Millipore).

    Techniques: Recombinant, In Vitro, SDS Page, Expressing, Purification, Molecular Weight, Affinity Chromatography