Journal: Macromolecular rapid communications

Article Title: Ultrasensitive Detection of Proteins on Western Blots with Semiconducting Polymer Dots

doi: 10.1002/marc.201200809

Figure Lengend Snippet: (A) Structure of CN-PPV polymer and the absorption (dashed) and emission (solid) spectra of CN-PPV Pdot (black) and CN-PPV Pdot-streptavidin conjugate (CNPPV-Strep, red) in a 0.1% PEG 20 mM HEPES buffer. (B) The absorption and emission spectra of Qdot

Article Snippet: To conjugate Pdots with streptavidin, to this 5 mL of Pdot solution, we added 100 μL of PEG (5 % w/v in water, MW 3350), 100 μL of 1 M HEPES buffer (pH 7.4), 300 μL of streptavidin (1 mg/mL in 20 mM HEPES buffer), and 50 μL of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, from Sigma-Aldrich) (10 mg/mL in water).

Techniques:

Journal: Molecular Genetics & Genomic Medicine

Article Title: Biochemical phenotype of a common disease-causing mutation and a possible therapeutic approach for the phosphomannomutase 2-associated disorder of glycosylation

doi: 10.1002/mgg3.3

Figure Lengend Snippet: Ligand binding can affect the thermal stability of PMM2. Heat-induced melting profile of wild-type PMM2 (A and C) and F119L-PMM2 (B and D) were recorded by thermal shift assay and by circular dichroism. For thermal shift assay, the proteins (0.2 mg/mL) were equilibrated in buffer (HEPES 20 mmol/L, NaCl 150 mmol/L, pH 7.5) containing Sypro Orange2.5X and the appropriate ligands: MgCl 2 5 mmol/L, EDTA 5 mmol/L, Glc-6-P 0.5 mmol/L + MgCl 2 5 mmol/L, Glc-6-P 0.5 mmol/L + MgCl 2 5 mmol/L + vanadate 0.5 mmol/L, Glu-1,6-P 0.5 mmol/L + MgCl 2 5 mmol/L. The samples were distributed in 96-well PCR plates, the plates were sealed, and heated from 25 to 80° at 0.5°C/min. The experiment was run on an iCycler iQ Real Time PCR Detection System. An excitation wavelength of 490 nm and an emission wavelength of 575 nm were used to collect the data. When the melting profile was obtained by circular dichroism the proteins (0.2 mg/mL) were equilibrated in the same buffer in the presence of MgCl 2 1 mmol/L or EDTA 5 mmol/L. The signal at 222 nm was recorded while temperature was increased at 0.5°C/min from 20°C. The raw data were corrected by taking into account the slopes of the pre- and post-transition baselines, then they were normalized.

Article Snippet: The proteins (0.2 mg/mL) were equilibrated in the presence of HEPES 20 mmol/L, NaCl 150 mmol/L, pH 7.5, Sypro Orange 2.5× (Invitrogen Molecular Probes, http://lifetechnologies.com ).

Techniques: Ligand Binding Assay, Thermal Shift Assay, Gas Chromatography, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

Journal: Molecular Genetics & Genomic Medicine

Article Title: Biochemical phenotype of a common disease-causing mutation and a possible therapeutic approach for the phosphomannomutase 2-associated disorder of glycosylation

doi: 10.1002/mgg3.3

Figure Lengend Snippet: Specific activity of PMM2 depends on glucose-1,6-bisphosphate concentration. The assay was performed at 32°C in a reaction mixture containing HEPES 20 mmol/L, pH 7.5, MgCl 2 5 mmol/L, NaCl 150 mmol/L, NADP+ 0.25 mmol/L, Glu-1-P (0.04 or 0.60 mmol/L), and yeast glucose 6-phosphate dehydrogenase 10 μg/mL, while Glc-1,6-P 2 was changed in the range 0–80 μmol/L. Enzymes concentrations were 107 nmol/L for wt-PMM2 and 73 nmol/L for F119L-PMM2. The hyperbolic dependence of velocity on the activator concentration was fitted using Michaelis and Menten equation to evaluate EC50.

Article Snippet: The proteins (0.2 mg/mL) were equilibrated in the presence of HEPES 20 mmol/L, NaCl 150 mmol/L, pH 7.5, Sypro Orange 2.5× (Invitrogen Molecular Probes, http://lifetechnologies.com ).

Techniques: Activity Assay, Concentration Assay, Gas Chromatography

Journal: Molecular Genetics & Genomic Medicine

Article Title: Biochemical phenotype of a common disease-causing mutation and a possible therapeutic approach for the phosphomannomutase 2-associated disorder of glycosylation

doi: 10.1002/mgg3.3

Figure Lengend Snippet: Long-term stability of F119L-PMM2. F119L-PMM2 (0.027 mmol/L of monomer equivalents) was equilibrated in HEPES 50 mmol/L pH 7.1 containing NaCl 150 mmol/L. Aliquots containing 1.6 μg of protein were taken at known incubation time and diluted immediately to assay the residual activity with Glc-1-P under standard conditions. (A) Results obtained at 37°C in the presence of EDTA 0.1 mmol/L or MgCl 2 5 mmol/L. (B) Results obtained at 44°C in the presence of MgCl 2 5 mmol/L, MgCl 2 5 mmol/L plus Glc-1-P 0.5 mmol/L, MgCl 2 5 mmol/L plus Glc-1-P 0.5 mmol/L and vanadate 0.5 mmol/L or MgCl 2 5 mmol/L plus Glu-1,6-P 2 0.5 mmol/L

Article Snippet: The proteins (0.2 mg/mL) were equilibrated in the presence of HEPES 20 mmol/L, NaCl 150 mmol/L, pH 7.5, Sypro Orange 2.5× (Invitrogen Molecular Probes, http://lifetechnologies.com ).

Techniques: Incubation, Activity Assay, Gas Chromatography

Journal: Molecular Genetics & Genomic Medicine

Article Title: Biochemical phenotype of a common disease-causing mutation and a possible therapeutic approach for the phosphomannomutase 2-associated disorder of glycosylation

doi: 10.1002/mgg3.3

Figure Lengend Snippet: Ligand binding can increase the resistance to proteases of PMM2. Purified wild-type PMM2 and F119L-PMM2 (A) were incubated (0.5 mg/mL) with thermolysin in HEPES 20 mmol/L, NaCl 150 mmol/L, MgCl 2 0.1 mmol/L, pH 7.5 at the indicated protease substrate ratio (w/w) for 1 or 2 h at 37°C before they were analyzed (5 μg of each sample) by SDS-PAGE. Purified F119L-PMM2 (B) was incubated (0.2 mg/mL) with thermolysin in HEPES 20 mmol/L, NaCl 150 mmol/L, MgCl 2 0.1 mmol/L, pH 7.5 at the indicated protease substrate ratio (w/w), in the presence of no ligands, Glc-1,6-P 2 0.5 mmol/L or Glu-6-P 0.5 mmol/L plus vanadate 0.5 mmol/L, for 2 h at 37°C before they were analyzed (2 μg of each sample) by SDS-PAGE. The protein bands were visualized by Coomassie blue staining and the intensity of the bands quantified. The not-digested protein was quantified and expressed as percentage of the starting material (no protease panel A; time 0 panel B).

Article Snippet: The proteins (0.2 mg/mL) were equilibrated in the presence of HEPES 20 mmol/L, NaCl 150 mmol/L, pH 7.5, Sypro Orange 2.5× (Invitrogen Molecular Probes, http://lifetechnologies.com ).

Techniques: Ligand Binding Assay, Purification, Incubation, SDS Page, Gas Chromatography, Staining

Journal: Molecular Genetics & Genomic Medicine

Article Title: Biochemical phenotype of a common disease-causing mutation and a possible therapeutic approach for the phosphomannomutase 2-associated disorder of glycosylation

doi: 10.1002/mgg3.3

Figure Lengend Snippet: Ligand binding affects the quaternary structure of PMM2. Wild-type PMM2 (0.010 mg) and F119L-PMM2 (0.0065 mg) were subjected to size exclusion chromatography on BioSep-SEC-S3000 column equilibrated in HEPES 20 mmol/L pH 7.5, NaCl 150 mmol/L, MgCl 2 5 mmol/L (long dashed line for the wild-type PMM2 or short dashed line for F119L-PMM2) or in the same buffer containing Glc-6-P 0.5 mmol/L and vanadate 0.1 mmol/L (continuous line for wild-type PMM2 or dotted line for F119L-PMM2). The chromatography was run at room temperature at 0.5 mL/min.

Article Snippet: The proteins (0.2 mg/mL) were equilibrated in the presence of HEPES 20 mmol/L, NaCl 150 mmol/L, pH 7.5, Sypro Orange 2.5× (Invitrogen Molecular Probes, http://lifetechnologies.com ).

Techniques: Ligand Binding Assay, Size-exclusion Chromatography, Gas Chromatography, Chromatography

Journal: Molecular Genetics & Genomic Medicine

Article Title: Biochemical phenotype of a common disease-causing mutation and a possible therapeutic approach for the phosphomannomutase 2-associated disorder of glycosylation

doi: 10.1002/mgg3.3

Figure Lengend Snippet: Specific activity of F119L-PMM2 depends on enzyme concentration. The assay was performed at 32°C in a reaction mixture containing HEPES 20 mmol/L, pH 7.5, MgCl 2 5 mmol/L, NaCl 150 mmol/L, NADP+ 0.25 mmol/L, Glc-1,6-P 2 0.030 mmol/L, and yeast glucose 6-phosphate dehydrogenase 10 μg/mL. The reaction mixture also contained BSA at 0.5 mg/mL. Three sets of experiments were carried out in the presence of 0.04, 0.16, or 0.6 mmol/L Glc-1-P and the F119L-PMM2 concentration changed in the range 10–240 nmol/L (monomer equivalents).

Article Snippet: The proteins (0.2 mg/mL) were equilibrated in the presence of HEPES 20 mmol/L, NaCl 150 mmol/L, pH 7.5, Sypro Orange 2.5× (Invitrogen Molecular Probes, http://lifetechnologies.com ).

Techniques: Activity Assay, Concentration Assay, Gas Chromatography

Journal: Molecular Genetics & Genomic Medicine

Article Title: Biochemical phenotype of a common disease-causing mutation and a possible therapeutic approach for the phosphomannomutase 2-associated disorder of glycosylation

doi: 10.1002/mgg3.3

Figure Lengend Snippet: Specific activity of wt-PMM2 changes as a function of protein concentration. The assay was performed at 32°C in a reaction mixture containing HEPES 20 mmol/L, pH 7.5, MgCl 2 5 mmol/L, NaCl 150 mmol/L, NADP+ 0.25 mmol/L, Glc-1,6-P 2 0.030 mmol/L and yeast glucose 6-phosphate dehydrogenase 0.010 mg/mL. The reaction mixture also contained Glc-1-P 0.020 mmol/L and BSA 0.5 mg/mL. The wt-PMM2 concentration changed in the range 2–110 nmol/L (monomer equivalents).

Article Snippet: The proteins (0.2 mg/mL) were equilibrated in the presence of HEPES 20 mmol/L, NaCl 150 mmol/L, pH 7.5, Sypro Orange 2.5× (Invitrogen Molecular Probes, http://lifetechnologies.com ).

Techniques: Activity Assay, Protein Concentration, Gas Chromatography, Concentration Assay

Journal: PLoS ONE

Article Title: Glutamate Uptake Triggers Transporter-Mediated GABA Release from Astrocytes

doi: 10.1371/journal.pone.0007153

Figure Lengend Snippet: Glu-induced GABA release is not mediated by vesicular release. (A) Elevation of [GABA] o in acute rat hippocampal slices in control conditions (n = 6) and in the presence of glutamate receptor antagonists (n = 7; P = 0.014; 128.5±13.4% of pre-stimulus control). (B) Cytosolic GABA level as determined by [ 3 H]GABA uptake in the presence of 100 µM NNC-711 and Glu receptor antagonists in rat cerebrocortical NPMV fractions (n = 4; P = 0.82) (C) Elevation of [GABA] o in acute rat hippocampal slices in control conditions (n = 6) and in low-[Ca 2+ ]/high-[Mg 2+ ] buffer (n = 5; P = 0.91; 148.3±15.5% of pre-stimulus control). (D) Cytosolic GABA level as determined by [ 3 H]GABA uptake in the presence of 100 µM NNC-711 in low-[Ca 2+ ] buffer in rat cerebrocortical NPMV fractions (n = 3; P = 0.49). Low-[Ca 2+ ] buffer contained 145 mM NaCl, 5 mM KCl, 20 mM MgCl 2 , 10 mM glucose and 20 mM HEPES (pH 7.5).

Article Snippet: Uptake studies were performed as follows: aliquots of NPMV fractions in HEPES buffer were preincubated with test compounds in the presence of NNC-711 (100 µM, Tocris) at 30°C for 10 min.

Techniques:

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Coptisine-induced inhibition of Helicobacter pylori: elucidation of specific mechanisms by probing urease active site and its maturation process

doi: 10.1080/14756366.2018.1501044

Figure Lengend Snippet: Ni 2+ and L-cys binding to coptisine (Cop) measured using UV-visible spectroscopy. 16 μM Cop in the absence or presence of different molar equivalents of Ni 2+ (1 mM NiCl 2 ) (A) or L-cys (1 mM) (B) in 20 mM HEPES buffer, pH 7.5. A-F, 0.0 to 320 μM of Ni 2+ ; A-F, 0.0 to 200 μM of L-cys. The inset shows the titration curves for binding of Ni 2+ or L-cys to Cop at 355 nm. Wavelength (nm) is represented on the x -axis and absorption (Abs) is represented on the y -axis.

Article Snippet: Acetohydroxamic acid (AHA; C2 H5 NO2 , purity: 98%), jack bean urease (JBU; type III with activity of 31.66 U/mg solid), urea, dithiothreitol (DTT), L-cysteine (L-cys), glutathione (GSH), sodium fluoride (NaF), and HEPES (Amresco > 99%) were purchased from Sigma Aldrich (Steineheim, Germany).

Techniques: Binding Assay, Spectroscopy, Titration

Journal: Clinical Proteomics

Article Title: Reproducible proteomics sample preparation for single FFPE tissue slices using acid-labile surfactant and direct trypsinization

doi: 10.1186/s12014-018-9188-y

Figure Lengend Snippet: Schematic Workflow for the DTR versus FASP comparison. Four single, deparaffinized murine kidney FFPE tissues were separately prepared with each of the three sample preparation protocol. For the DTR protocol, a buffer containing 0.1% Rapigest in 0.1 M HEPES pH 8 and 1 mM DTT was used for heat-induced antigen retrieval (HIAR) and lysis of the tissue. As RapiGest is compatible with tryptic digestion, direct trypsinization is the key feature of the DTR protocol. RapiGest is later removed by acidifying the sample. Protein concentration is estimated to not overload the C18 stage tips during desalting step and later to inject the same amounts of peptides into the mass spectrometer. The FASP protocol makes use of a buffer containing 4% SDS, 0.1 M HEPES pH 7.5 and 0.05 M DTT. Before digestion the SDS is removed using centrifugal filter units with nominal molecular weight cut offs of either 10,000 or 30,000 Da. After digestion, the peptides are eluted from the filter units and desalted before mass-spectrometry analysis

Article Snippet: 100 µl (200 µl for the tonsil samples) of an aqueous buffer containing 0.1% RapiGest SF (Waters, Milford, MA, USA), 0.1 M HEPES pH 8 (AppliChem, Darmstadt, Germany) and 1 mM dithiothreitol (DTT) (AppliChem, Darmstadt, Germany) were added to each reaction tube, containing a single tissue slice.

Techniques: Formalin-fixed Paraffin-Embedded, Sample Prep, Lysis, Protein Concentration, Mass Spectrometry, Molecular Weight

Journal: Pharmaceutical Research

Article Title: Tuning Ciprofloxacin Release Profiles from Liposomally Encapsulated Nanocrystalline Drug

doi: 10.1007/s11095-016-2002-5

Figure Lengend Snippet: Evaluation of the effect of freeze-thaw at −50°C on the IVR profiles of CFI formulations at pH 6.0 containing 90 mg/ml sucrose and polysorbate 20 after freeze-thaw. The CFI formulations (at 12.5 mg/ml) were diluted to 50 μg/ml ciprofloxacin in HEPES buffered saline (HBS) prior to a 1:1 dilution in bovine serum to measure the release of ciprofloxacin after incubation at 37°C for up to 4 h. IVR profiles are shown for the CFI control (no freeze-thaw, blue diamonds), CFI containing 0.05% polysorbate 20 after freeze-thaw (red squares), CFI containing 0.1% polysorbate 20 after freeze-thaw (green triangles), and CFI containing 0.2% polysorbate 20 after freeze-thaw (yellow circles). Duplicate samples were analyzed at each time point.

Article Snippet: The following materials were used for the preparation, characterization or analysis of the liposomal ciprofloxacin (CFI) formulations: sucrose (Sigma-Aldrich (St. Louis, MO)), HEPES, free acid (Avantor (Center Valley, PA)), sodium chloride (Amresco (Solon, OH)), HPLC grade methanol (Fisher Scientific (Fair Lawn, NJ)), triethylamine (TEA, JT Baker (USA)), polysorbate 20 (VWR Int. (West Chester, NJ)), polyethylene glycol dodecyl ether (Brij 30 (Sigma-Aldrich (Australia))), Donor Adult Bovine Serum (HyClone (Logan, Utah)), and Nanosep centrifugal filtration devices, 10 K and 30 K molecular weight (Pall Corporation (Ann Arbor, MI)).

Techniques: Incubation

Journal: Pharmaceutical Research

Article Title: Tuning Ciprofloxacin Release Profiles from Liposomally Encapsulated Nanocrystalline Drug

doi: 10.1007/s11095-016-2002-5

Figure Lengend Snippet: Evaluation of the effect of freeze-thaw in liquid nitrogen on the IVR profiles of CFI formulations at pH 6.0 containing 90 mg/ml sucrose and 0, 0.05 or 0.1% polysorbate 20 after freeze-thaw. The CFI formulations (at 12.5 mg/ml) were diluted to 50 μg/ml ciprofloxacin in HEPES buffered saline (HBS) prior to a 1:1 dilution in bovine serum to measure the release of ciprofloxacin after incubation at 37°C for up to 4 h. IVR profiles are shown for the CFI control prior to freeze-thaw ( blue diamonds ) or after freeze-thaw ( red circles ), CFI containing 0.05% polysorbate 20 after freeze-thaw ( green diamonds ), CFI containing 0.1% polysorbate 20 after freeze-thaw ( yellow triangles ). Duplicate samples were analyzed at each time point. Two simulated curves were generated by adjusting the release from the CFI sample without polysorbate 20 after freeze-thaw by assuming the addition of either 10% free drug ( green dashed line ) or 20% free drug ( yellow dashed line ) and normalizing the total drug release to 100%.

Article Snippet: The following materials were used for the preparation, characterization or analysis of the liposomal ciprofloxacin (CFI) formulations: sucrose (Sigma-Aldrich (St. Louis, MO)), HEPES, free acid (Avantor (Center Valley, PA)), sodium chloride (Amresco (Solon, OH)), HPLC grade methanol (Fisher Scientific (Fair Lawn, NJ)), triethylamine (TEA, JT Baker (USA)), polysorbate 20 (VWR Int. (West Chester, NJ)), polyethylene glycol dodecyl ether (Brij 30 (Sigma-Aldrich (Australia))), Donor Adult Bovine Serum (HyClone (Logan, Utah)), and Nanosep centrifugal filtration devices, 10 K and 30 K molecular weight (Pall Corporation (Ann Arbor, MI)).

Techniques: Incubation, Generated

Journal: Pharmaceutical Research

Article Title: Tuning Ciprofloxacin Release Profiles from Liposomally Encapsulated Nanocrystalline Drug

doi: 10.1007/s11095-016-2002-5

Figure Lengend Snippet: Evaluation of the effect of freeze-thaw at −50°C on the IVR profiles of CFI formulations at pH 6.0 containing 90 mg/ml sucrose and Brij 30 before ( a ) and after ( b ) freeze-thaw. The 12.5 mg/ml CFI formulations were diluted to 50 μg/ml ciprofloxacin in HEPES buffered saline (HBS) prior to a 1:1 dilution in bovine serum to measure the release of ciprofloxacin after incubation at 37°C for up to 4 h. IVR profiles are shown for the CFI control (no freeze thaw, blue diamonds ), CFI containing 0.05% Brij 30 ( red squares ), CFI containing 0.1% Brij 30 ( green triangles ), CFI containing 0.2% Brij 30 ( orange circles ), and CFI containing 0.3% Brij 30 ( purple squares ). Duplicate samples were analyzed at each time point.

Article Snippet: The following materials were used for the preparation, characterization or analysis of the liposomal ciprofloxacin (CFI) formulations: sucrose (Sigma-Aldrich (St. Louis, MO)), HEPES, free acid (Avantor (Center Valley, PA)), sodium chloride (Amresco (Solon, OH)), HPLC grade methanol (Fisher Scientific (Fair Lawn, NJ)), triethylamine (TEA, JT Baker (USA)), polysorbate 20 (VWR Int. (West Chester, NJ)), polyethylene glycol dodecyl ether (Brij 30 (Sigma-Aldrich (Australia))), Donor Adult Bovine Serum (HyClone (Logan, Utah)), and Nanosep centrifugal filtration devices, 10 K and 30 K molecular weight (Pall Corporation (Ann Arbor, MI)).

Techniques: Incubation

Journal: Frontiers in Immunology

Article Title: Serum and Serum Albumin Inhibit in vitro Formation of Neutrophil Extracellular Traps (NETs)

doi: 10.3389/fimmu.2019.00012

Figure Lengend Snippet: Extracellular NETotic DNA is rich in MPO. Representative fluorescence images of NETs induced by CaI (4 μM), PMA (100 nM), or LPS (25 μg/ml) activated in supplement-free RPMI/HEPES. The images show a clear colocalization of MPO (red) with the extracellular DNA-fibers (blue) of released NETs. Scale = 10 μm.

Article Snippet: Freshly isolated human or murine neutrophils were counted and suspended in RPMI 1640 (Lonza) containing 10 mM HEPES (Roth) (RPMI/HEPES) and 0.5% HSA (Sigma-Aldrich), 0.5%/1%/2% FCS (Biochrom GmbH, Merck Millipore) or 0.5% BSA (Roth), respectively.

Techniques: Fluorescence

Journal: Frontiers in Immunology

Article Title: Serum and Serum Albumin Inhibit in vitro Formation of Neutrophil Extracellular Traps (NETs)

doi: 10.3389/fimmu.2019.00012

Figure Lengend Snippet: Influence of serum and serum albumin supplements on NET formation of murine neutrophils. (A) Representative fluorescence images of nuclei of murine neutrophils (Hoechst) after stimulation with CaI (4 μM), PMA (100 nM) or LPS (25 μg/ml) for 180 min, respectively. NET formation of neutrophils was studied in RPMI/ HEPES without supplements and RPMI/ HEPES supplemented with 0.5% BSA or 2% hiFCS, as indicated. Chromatin decondensation is only inducible in RPMI/ HEPES (white arrow heads). Scale = 50 μm. (B) Percentage of decondensed nuclei/ NETs after stimulation with PMA (100 nM), CaI (4 μM), or LPS (10, 25, or 100 μg/ml) for 180 min. Murine neutrophils were studied in RPMI/ HEPES and RPMI/ HEPES supplemented with 0.5% BSA or 2% hiFCS. Addition of 0.5% BSA or 2% hiFCS to RPMI/ HEPES, inhibits chromatin decondensation induced by PMA, CaI and LPS completely. Error bars = mean ± SEM. ns = not significant. **** p

Article Snippet: Freshly isolated human or murine neutrophils were counted and suspended in RPMI 1640 (Lonza) containing 10 mM HEPES (Roth) (RPMI/HEPES) and 0.5% HSA (Sigma-Aldrich), 0.5%/1%/2% FCS (Biochrom GmbH, Merck Millipore) or 0.5% BSA (Roth), respectively.

Techniques: Fluorescence

Journal: Frontiers in Immunology

Article Title: Serum and Serum Albumin Inhibit in vitro Formation of Neutrophil Extracellular Traps (NETs)

doi: 10.3389/fimmu.2019.00012

Figure Lengend Snippet: Influence of serum and serum albumin supplements on NET formation of human neutrophils. (A) Representative fluorescence images of human neutrophils (chromatin stained by Hoechst) after stimulation with CaI (4 μM), PMA (100 nM), or LPS (25 μg/ml) for 180 min, respectively. NET formation of neutrophils was studied in RPMI 1,640 with 10 mM HEPES (RPMI/HEPES), RPMI/ HEPES + 0.5% human serum albumin (HSA) or + 1% heat inactivated (56°C) fetal calf serum (hiFCS). Chromatin decondensation induced by PMA is clearly visible with all three culture conditions, while LPS or CaI only cause NET formation in BSA- and HSA-free RPMI/HEPES. Scale = 50 μm. (B) Neutrophils were stimulated to undergo NET formation with CaI (4 μM), PMA (100 nM) or LPS (10, 25, or 100 μg/ml), respectively. Both, HSA and BSA inhibit CaI and LPS-induced formation of NETs (determined as percentage of decondensed nuclei/NETs of total neutrophils). NETosis stimulated by PMA is independent of serum albumin addition. Error bars = mean ± SEM. ns, not significant. * p

Article Snippet: Freshly isolated human or murine neutrophils were counted and suspended in RPMI 1640 (Lonza) containing 10 mM HEPES (Roth) (RPMI/HEPES) and 0.5% HSA (Sigma-Aldrich), 0.5%/1%/2% FCS (Biochrom GmbH, Merck Millipore) or 0.5% BSA (Roth), respectively.

Techniques: Fluorescence, Staining, Albumin Addition

Journal: Biochemistry

Article Title: Electrostatic and Hydrophobic Interactions Mediate Single-Stranded DNA Recognition and Acta2 Repression by Purine-Rich Element Binding Protein B

doi: 10.1021/acs.biochem.6b00006

Figure Lengend Snippet: Effect of solution pH on the interaction of Purβ with Acta2 ssDNA. (A) The binding of full-length Purβ (1.0 nM) to PE32-bF ssDNA (0.5 nM) was assessed in assay buffer without MgCl 2 at pH ranging from 7.5 to 12.5. Solid-phase Purβ-PE32-bF complexes were detected by ELISA using rabbit anti-mouse Purβ 210–229 as the primary antibody. Nonspecific background absorbance at 405 nm in control wells without any DNA was subtracted from the signal generated in PE32-bF-coated wells. Background corrected A405 values measured at each pH were normalized by dividing by the mean A405 value determined at pH 7.5 (mean ± SD, n = 4). (B) Effect of solution pH on the thermostability of Purβ. The unfolding of full-length Purβ was evaulated by thermal shift assay at a protein concentration of 2.8 μM in 20 mM HEPES, 150 mM NaCl, 10 mM β-mercaptoethanol adjusted to pH 7.5, 10.5, 11.5, or 12.5.

Article Snippet: Briefly, NHis-tagged MSY1 was diluted to 100 nM in coating buffer consisting of 20 mM HEPES, 150 mM NaCl, 1.5 mM MgCl2 -6H2 O pH 7.5 with 5.0 μg/ml BSA and then applied to microtiter wells (Costar® EIA/RIA plate, 96 Well Easy Wash™, Certified High Binding, Corning, Inc.).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Generated, Thermal Shift Assay, Protein Concentration

Journal: Planta

Article Title: Biosynthesis of the carbohydrate moieties of arabinogalactan proteins by membrane-bound ?-glucuronosyltransferases from radish primary roots

doi: 10.1007/s00425-013-1959-0

Figure Lengend Snippet: Activity of β-GlcATs in microsomes from radish primary roots. a The microsomes (protein, 50 μg) were incubated under standard assay conditions with β-(1 → 3)-galactan as exogenous acceptor ( closed circles ) and with endogenous acceptors ( open circles ) for varying times up to 240 min. After 240 min, the amount of [ 14 C]GlcA transferred to β-(1 → 3)-galactan was about 0.6 % of initial UDP-[ 14 C]GlcA. The data are the mean + SE of quadruple experiments. b Relationship between enzyme activity and protein concentration. Various amounts of microsomal protein were incubated under standard conditions for 60 min. c Effects of pH on enzyme activity. Activity–pH curves result from experiments with buffers (80 mM) having different pH values under standard conditions for 120 min. Closed circles Mes–KOH, open circles Hepes–KOH, closed triangles acetate. d Activity–temperature curves result from incubations at different temperatures under standard assay conditions for 120 min. Note that the activity plotted for β-(1 → 3)-galactan includes that for endogenous acceptors in a and b

Article Snippet: EGTA, Hepes, and Mes were from Dojindo Laboratories (Kumamoto, Japan). β-(1 → 3)- and β-(1 → 6)-Galactobioses, and -trioses were prepared from larch wood AG by partial acid hydrolysis (Aspinall et al. ).

Techniques: Activity Assay, Incubation, Protein Concentration

Journal: Pharmaceutical research

Article Title: Protected Graft Copolymer Excipient Leads to a Higher Acute Maximum Tolerated Dose and Extends Residence Time of Vasoactive Intestinal Peptide Significantly Better than Sterically Stabilized Micelles

doi: 10.1007/s11095-012-0904-4

Figure Lengend Snippet: VIP binding in 100 mM NaCl, 100 mM HEPES at a carrier concentration of 2.4 mg/ml for PGC and 2.0 mg/ml for SSM ( a ) PGC-VIP, ( b ) SSM-VIP. The experiment was done in quadruplicate; free VIP was separated from bound by ultrafiltration, VIP quantitation was

Article Snippet: VIP was mixed with a fixed amount of PGC (0.6 mg at 2.4 mg/ml final concentration) or SSM carriers (0.5 mg at 2 mg/ml final concentration) at concentrations between 72 μM and 188 μM in the PGC mixtures and between 120 μM and 240 μM in the SSM mixtures in an aqueous solution of 100 mM NaCl (Fisher Scientific, Waltham MA) and 100 mM HEPES (Fisher Scientific, Waltham MA) in quadruplicate.

Techniques: Binding Assay, Concentration Assay, Pyrolysis Gas Chromatography, Quantitation Assay