tris  (Millipore)


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    Tris
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    Structured Review

    Millipore tris
    Tris

    https://www.bioz.com/result/tris/product/Millipore
    Average 99 stars, based on 2261 article reviews
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    tris - by Bioz Stars, 2020-09
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    Images

    1) Product Images from "Glycoinositolphospholipids from Trypanosomatids Subvert Nitric Oxide Production in Rhodnius prolixus Salivary Glands"

    Article Title: Glycoinositolphospholipids from Trypanosomatids Subvert Nitric Oxide Production in Rhodnius prolixus Salivary Glands

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0047285

    NADPH-diaphorase activity of NOS in Rhodnius prolixus salivary glands after a blood meal and the expression of NOS. A. Salivary glands were dissected in different days after blood feeding and evaluated for NOS NAPDH-diaphorase activity. Salivary glands were assayed in 10 mM Tris-HCl pH 8,0, 0,05 M NaCl, 0,1%, Triton X-100, 1 mM CaCl 2 , 5 µM FAD, 1 mM NADPH and 0,5 mg/mL MTT. MTT reduction was followed at 540 nm for 30 min at 37°C. Also samples were obtained and NOS content evaluated by Western blotting. Each point is the average and SE of 05 different experiments. B. Immunoblotting using an anti-NOS antibody. Blottings were developed with the use of a secondary antibody conjugated to alkaline phosphatase in the presence of the substrate Western Blue. Molecular mass markers are indicated at the left. C. Upper panel , total RNA from the salivary glands at different days after feeding was isolated and cDNA was synthesized. Samples were then analyzed by semi-quantitative PCR with temperatures of 55, 72 and 94°C for 27 cycles with primers for NOS. Lower panel, analysis of 18 S RNA levels. In this case reaction occurred for 25 cycles. The products of reactions shown on panels C were separated on agarose gel 1.4% stained with ethidium bromide and photographed under ultraviolet light. Molecular mass standards are indicated at the left.
    Figure Legend Snippet: NADPH-diaphorase activity of NOS in Rhodnius prolixus salivary glands after a blood meal and the expression of NOS. A. Salivary glands were dissected in different days after blood feeding and evaluated for NOS NAPDH-diaphorase activity. Salivary glands were assayed in 10 mM Tris-HCl pH 8,0, 0,05 M NaCl, 0,1%, Triton X-100, 1 mM CaCl 2 , 5 µM FAD, 1 mM NADPH and 0,5 mg/mL MTT. MTT reduction was followed at 540 nm for 30 min at 37°C. Also samples were obtained and NOS content evaluated by Western blotting. Each point is the average and SE of 05 different experiments. B. Immunoblotting using an anti-NOS antibody. Blottings were developed with the use of a secondary antibody conjugated to alkaline phosphatase in the presence of the substrate Western Blue. Molecular mass markers are indicated at the left. C. Upper panel , total RNA from the salivary glands at different days after feeding was isolated and cDNA was synthesized. Samples were then analyzed by semi-quantitative PCR with temperatures of 55, 72 and 94°C for 27 cycles with primers for NOS. Lower panel, analysis of 18 S RNA levels. In this case reaction occurred for 25 cycles. The products of reactions shown on panels C were separated on agarose gel 1.4% stained with ethidium bromide and photographed under ultraviolet light. Molecular mass standards are indicated at the left.

    Techniques Used: Activity Assay, Expressing, MTT Assay, Western Blot, Isolation, Synthesized, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining

    2) Product Images from "Simple buffers for 3D STORM microscopy"

    Article Title: Simple buffers for 3D STORM microscopy

    Journal: Biomedical Optics Express

    doi: 10.1364/BOE.4.000885

    Statistics on STORM imaging performed in 25% Vectashield - 75% TRIS-Glycerol in which were added 1% NPG (w/v) (A), 20 mM DABCO (B), and 10 mM Lipoic Acid (C). The different panels represent: (1) photon count distribution per frame and per molecule, averaged over three datasets, (2) standard deviation of multiple localizations giving a measure of the frame localization precision, and (3) Density of molecules as a function of number of recorded frames, averaged over three measurements, with error bars indicating the standard deviation.
    Figure Legend Snippet: Statistics on STORM imaging performed in 25% Vectashield - 75% TRIS-Glycerol in which were added 1% NPG (w/v) (A), 20 mM DABCO (B), and 10 mM Lipoic Acid (C). The different panels represent: (1) photon count distribution per frame and per molecule, averaged over three datasets, (2) standard deviation of multiple localizations giving a measure of the frame localization precision, and (3) Density of molecules as a function of number of recorded frames, averaged over three measurements, with error bars indicating the standard deviation.

    Techniques Used: Imaging, Standard Deviation

    3) Product Images from "Identification of Small-Molecule Inhibitors of Yersinia pestis Type III Secretion System YscN ATPase"

    Article Title: Identification of Small-Molecule Inhibitors of Yersinia pestis Type III Secretion System YscN ATPase

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0019716

    Steady-state kinetics of ATP hydrolysis by the optimized catalytic domain. (A) The ATP hydrolysis is inhibited above 4 mM ATP concentration. (B) The hydrolysis of ATP by the enzyme shows a positive cooperativity up to 4 mM ATP concentration. The kinetics of hydrolysis was measured by following phosphate release in 10 mM Tris, pH = 7.6, 150 mM NaCl, and 1 mM Mg +2 at 37°C as described under Materials and Methods . Total protein concentration was 9.6 µg. Error bars correspond to standard deviation of triplicate measurements.
    Figure Legend Snippet: Steady-state kinetics of ATP hydrolysis by the optimized catalytic domain. (A) The ATP hydrolysis is inhibited above 4 mM ATP concentration. (B) The hydrolysis of ATP by the enzyme shows a positive cooperativity up to 4 mM ATP concentration. The kinetics of hydrolysis was measured by following phosphate release in 10 mM Tris, pH = 7.6, 150 mM NaCl, and 1 mM Mg +2 at 37°C as described under Materials and Methods . Total protein concentration was 9.6 µg. Error bars correspond to standard deviation of triplicate measurements.

    Techniques Used: Concentration Assay, Protein Concentration, Standard Deviation

    4) Product Images from "Bacterially Expressed F1-20/AP-3 Assembles Clathrin Into Cages With a Narrow Size Distribution: Implications for the Regulation of Quantal Size During Neurotransmission"

    Article Title: Bacterially Expressed F1-20/AP-3 Assembles Clathrin Into Cages With a Narrow Size Distribution: Implications for the Regulation of Quantal Size During Neurotransmission

    Journal: Journal of neuroscience research

    doi: 10.1002/jnr.490410104

    The bacterially expressed 33 kD NH 2 -terminus of F1-20/AP-3 binds specifically to clathrin triskelia; 15 µg of the bacterially expressed 33 kD NH 2 -terminus of F1-20/AP-3 was incubated with 0.5 ml clathrin-Sepharose in 0.5 ml isolation buffer at 4°C for 2 hr ( A ), and binding was monitored by batch analysis, as described in Methods. Fraction 1 is the flow-through; fractions 2,3,4 are washes with isolation buffer; and fractions 5,6,7 are eluates with 0.5 M Tris (pH 7.0). All samples were analyzed by SDS-PAGE, followed by silver staining. Negative controls were carried out by incubating 15 µg bacterially expressed 33 kD NH 2 -terminus of F1-20/AP-3 with 0.5 ml underivatized Sepharose ( B ), and by incubating 15 µg E. coli GST protein with 0.5 ml clathrin-Sepharose ( C ).
    Figure Legend Snippet: The bacterially expressed 33 kD NH 2 -terminus of F1-20/AP-3 binds specifically to clathrin triskelia; 15 µg of the bacterially expressed 33 kD NH 2 -terminus of F1-20/AP-3 was incubated with 0.5 ml clathrin-Sepharose in 0.5 ml isolation buffer at 4°C for 2 hr ( A ), and binding was monitored by batch analysis, as described in Methods. Fraction 1 is the flow-through; fractions 2,3,4 are washes with isolation buffer; and fractions 5,6,7 are eluates with 0.5 M Tris (pH 7.0). All samples were analyzed by SDS-PAGE, followed by silver staining. Negative controls were carried out by incubating 15 µg bacterially expressed 33 kD NH 2 -terminus of F1-20/AP-3 with 0.5 ml underivatized Sepharose ( B ), and by incubating 15 µg E. coli GST protein with 0.5 ml clathrin-Sepharose ( C ).

    Techniques Used: Incubation, Isolation, Binding Assay, Flow Cytometry, SDS Page, Silver Staining

    5) Product Images from "Cj1386, an Atypical Hemin-Binding Protein, Mediates Hemin Trafficking to KatA in Campylobacter jejuni"

    Article Title: Cj1386, an Atypical Hemin-Binding Protein, Mediates Hemin Trafficking to KatA in Campylobacter jejuni

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.02346-14

    Tyrosine 57 is important for hemin affinity to Cj1386. (A) Absorption spectra of 10 μM Cj1386 WT and 10 μM Cj1386 Y57A in 100 mM NaCl, 20 mM Tris, pH 7.4. (B) Absorption spectra of 10 μM Cj1386 Y57A and 10 μM Cj1386 Y57A plus
    Figure Legend Snippet: Tyrosine 57 is important for hemin affinity to Cj1386. (A) Absorption spectra of 10 μM Cj1386 WT and 10 μM Cj1386 Y57A in 100 mM NaCl, 20 mM Tris, pH 7.4. (B) Absorption spectra of 10 μM Cj1386 Y57A and 10 μM Cj1386 Y57A plus

    Techniques Used:

    Y57A Cj1386 can be reconstituted with hemin and displays 1:1 hemin-binding stoichiometry. (A) Absorption spectra of Cj1386 Y57A when titrated with hemin at 1 μM increments against 10 μM apo-Cj1386 Y57A in 100 mM NaCl, 20 mM Tris, pH 7.4.
    Figure Legend Snippet: Y57A Cj1386 can be reconstituted with hemin and displays 1:1 hemin-binding stoichiometry. (A) Absorption spectra of Cj1386 Y57A when titrated with hemin at 1 μM increments against 10 μM apo-Cj1386 Y57A in 100 mM NaCl, 20 mM Tris, pH 7.4.

    Techniques Used: Binding Assay

    6) Product Images from "Quantum and electrochemical interplays in hydrogenated graphene"

    Article Title: Quantum and electrochemical interplays in hydrogenated graphene

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03026-0

    Transport characteristic and quantum capacitance of CVD graphene upon hydrogenation. a Illustration of the field effect transistor setup fabricated from CVD graphene. b Room temperature conductance ( G ) plots as a function of the gate voltage ( V g ) showing the p-doping effect upon hydrogenation from 0 to 30 s. The gray dashed line is a guide-to-the-eye, highlighting the sublinear behavior of the G ( V g ) curves. c The shifts of the charge neutrality point (CNP) upon hydrogenation. d The carrier mobility of graphene, µ , vs the hydrogenation time. e Quantum capacitance C q of graphene measured as a function of V ch for 0–30 s of hydrogenation. f Impurity density n imp vs hydrogenation time. The electrolyte solution is 0.1 M KCl with 10 mM Tris (pH 8). The error bars in d , f are the standard deviation of experimental values
    Figure Legend Snippet: Transport characteristic and quantum capacitance of CVD graphene upon hydrogenation. a Illustration of the field effect transistor setup fabricated from CVD graphene. b Room temperature conductance ( G ) plots as a function of the gate voltage ( V g ) showing the p-doping effect upon hydrogenation from 0 to 30 s. The gray dashed line is a guide-to-the-eye, highlighting the sublinear behavior of the G ( V g ) curves. c The shifts of the charge neutrality point (CNP) upon hydrogenation. d The carrier mobility of graphene, µ , vs the hydrogenation time. e Quantum capacitance C q of graphene measured as a function of V ch for 0–30 s of hydrogenation. f Impurity density n imp vs hydrogenation time. The electrolyte solution is 0.1 M KCl with 10 mM Tris (pH 8). The error bars in d , f are the standard deviation of experimental values

    Techniques Used: Standard Deviation

    Electrochemical behavior of CVD graphene upon hydrogenation. a Cyclic voltammograms (CVs) obtained on graphene after 0–30 s of hydrogenation at a scan rate of 100 mV s −1 . b Current density vs scan rate for untreated graphene shown in a . c The electron transfer rate k 0 vs hydrogenation time from 0 to 30 s. d The averaged total capacitance C ave− tot vs hydrogenation time from 0 to 30 s. e CV curves obtained on graphene after 0–13 s of Ar treatment at a scan rate of 100 mV s −1 . f \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$k^0_{{\mathrm{Ar}}}$$\end{document} k Ar 0 vs argon plasma treating time from 0 to 13 s. The aqueous electrolyte solution contains 0.1 M KCl supplemented with 10 mM Tris at pH 8. The redox probe employed is 1 mM hexaammineruthenium (II)/hexaammineruthenium (III) chloride. The error bars in c , d , f are the standard deviation of experimental values
    Figure Legend Snippet: Electrochemical behavior of CVD graphene upon hydrogenation. a Cyclic voltammograms (CVs) obtained on graphene after 0–30 s of hydrogenation at a scan rate of 100 mV s −1 . b Current density vs scan rate for untreated graphene shown in a . c The electron transfer rate k 0 vs hydrogenation time from 0 to 30 s. d The averaged total capacitance C ave− tot vs hydrogenation time from 0 to 30 s. e CV curves obtained on graphene after 0–13 s of Ar treatment at a scan rate of 100 mV s −1 . f \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$k^0_{{\mathrm{Ar}}}$$\end{document} k Ar 0 vs argon plasma treating time from 0 to 13 s. The aqueous electrolyte solution contains 0.1 M KCl supplemented with 10 mM Tris at pH 8. The redox probe employed is 1 mM hexaammineruthenium (II)/hexaammineruthenium (III) chloride. The error bars in c , d , f are the standard deviation of experimental values

    Techniques Used: Standard Deviation

    7) Product Images from "Influence of sodium substitutes on 5-HT-mediated effects at mouse 5-HT3 receptors"

    Article Title: Influence of sodium substitutes on 5-HT-mediated effects at mouse 5-HT3 receptors

    Journal: British Journal of Pharmacology

    doi: 10.1038/sj.bjp.0705788

    Chemical structures of the organic cations used in this study to replace sodium. NMDG (pka=9.5) and Tris (pka=8.3) are positively charged at physiological pH. The ion diameters (Angstrom units, three dimensions) are as follows: sodium 1.924, 1.924 and 1.924; choline 7.487, 5.020 and 4.488; guanidinium 6.498, 4.952 and 3.103; NMDG 13.056, 5.582 and 5.332; TEA 8.431, 6.358 and 5.841; TMA 5.291, 4.824 and 4.556; and Tris 7.644, 5.712 and 4.491. The values for lithium are 1.198, 1.198 and 1.198.
    Figure Legend Snippet: Chemical structures of the organic cations used in this study to replace sodium. NMDG (pka=9.5) and Tris (pka=8.3) are positively charged at physiological pH. The ion diameters (Angstrom units, three dimensions) are as follows: sodium 1.924, 1.924 and 1.924; choline 7.487, 5.020 and 4.488; guanidinium 6.498, 4.952 and 3.103; NMDG 13.056, 5.582 and 5.332; TEA 8.431, 6.358 and 5.841; TMA 5.291, 4.824 and 4.556; and Tris 7.644, 5.712 and 4.491. The values for lithium are 1.198, 1.198 and 1.198.

    Techniques Used:

    8) Product Images from "Effect of Common Buffers and Heterocyclic Ligands on the Binding of Cu(II) at the Multimetal Binding Site in Human Serum Albumin"

    Article Title: Effect of Common Buffers and Heterocyclic Ligands on the Binding of Cu(II) at the Multimetal Binding Site in Human Serum Albumin

    Journal: Bioinorganic Chemistry and Applications

    doi: 10.1155/2010/725153

    Titration curves (experimental points, , and fits to the K app formula, ) for Cu(II) binding at MBS in four solutions studied: (a) NaCl; (b) Hepes; (c) sodium phosphate; (d) Tris.
    Figure Legend Snippet: Titration curves (experimental points, , and fits to the K app formula, ) for Cu(II) binding at MBS in four solutions studied: (a) NaCl; (b) Hepes; (c) sodium phosphate; (d) Tris.

    Techniques Used: Titration, Binding Assay

    9) Product Images from "Amyloid-Like Aggregates of the Yeast Prion Protein Ure2 Enter Vertebrate Cells by Specific Endocytotic Pathways and Induce Apoptosis"

    Article Title: Amyloid-Like Aggregates of the Yeast Prion Protein Ure2 Enter Vertebrate Cells by Specific Endocytotic Pathways and Induce Apoptosis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0012529

    Formation of amyloid-like structure for Ure2. Fibril formation was monitored by binding of the fluorescent dye ThT, AFM and CD. ( A ) Kinetics of formation of amyloid-like structure for Ure2 monitored by ThT binding. ( B ) Far-UV CD spectra of 20 µM native Ure2 and protofibrils in 50 mM Tris-HCl buffer containing 0.2 M NaCl (pH 7.5). 20 µM Native Ure2 (−), 20 µM protofibrils of Ure2 (…). ( C ), ( D ) Morphology of amyloid-like structures monitored by AFM for WT Ure2. ( C ) Protofibrils (height 3–10 nm) were abundant at early exponential growth phase of the fibril growth curve. ( D ) Mature fibrils (height 12–15 nm) were predominant at later time points (plateau phase).
    Figure Legend Snippet: Formation of amyloid-like structure for Ure2. Fibril formation was monitored by binding of the fluorescent dye ThT, AFM and CD. ( A ) Kinetics of formation of amyloid-like structure for Ure2 monitored by ThT binding. ( B ) Far-UV CD spectra of 20 µM native Ure2 and protofibrils in 50 mM Tris-HCl buffer containing 0.2 M NaCl (pH 7.5). 20 µM Native Ure2 (−), 20 µM protofibrils of Ure2 (…). ( C ), ( D ) Morphology of amyloid-like structures monitored by AFM for WT Ure2. ( C ) Protofibrils (height 3–10 nm) were abundant at early exponential growth phase of the fibril growth curve. ( D ) Mature fibrils (height 12–15 nm) were predominant at later time points (plateau phase).

    Techniques Used: Binding Assay

    10) Product Images from "Glyco-engineered Long Acting FGF21 Variant with Optimal Pharmaceutical and Pharmacokinetic Properties to Enable Weekly to Twice Monthly Subcutaneous Dosing"

    Article Title: Glyco-engineered Long Acting FGF21 Variant with Optimal Pharmaceutical and Pharmacokinetic Properties to Enable Weekly to Twice Monthly Subcutaneous Dosing

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-22456-w

    Formulation characterization of PF-06645849. ( a ) % HMMS formation at 25 °C in Tris pH 7.5 formulation for WT FGF21 (40 mg/mL), Fc-FGF21[N171] (100 mg/mL) and PF-06645849/Fc-FGF21 [R19V][N171] (100 mg/mL) over time. ( b ) % HMMS formation for PF-06645849 at pH 7.5/25 °C over time at concentrations of 61, 82, and 100 mg/mL, respectively. ( c ) Viscosity of PF-06645849/Fc-FGF21[R19V][N171] over concentration ranges. All experiments were carried out as n = 1.
    Figure Legend Snippet: Formulation characterization of PF-06645849. ( a ) % HMMS formation at 25 °C in Tris pH 7.5 formulation for WT FGF21 (40 mg/mL), Fc-FGF21[N171] (100 mg/mL) and PF-06645849/Fc-FGF21 [R19V][N171] (100 mg/mL) over time. ( b ) % HMMS formation for PF-06645849 at pH 7.5/25 °C over time at concentrations of 61, 82, and 100 mg/mL, respectively. ( c ) Viscosity of PF-06645849/Fc-FGF21[R19V][N171] over concentration ranges. All experiments were carried out as n = 1.

    Techniques Used: Concentration Assay

    11) Product Images from "Non-uniform self-assembly: On the anisotropic architecture of α-synuclein supra-fibrillar aggregates"

    Article Title: Non-uniform self-assembly: On the anisotropic architecture of α-synuclein supra-fibrillar aggregates

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-06532-1

    Alignment of αS SFAs at oil/water interface. ( A ) Ratiometric confocal fluorescence image of an αS SFA containing aS140C-MFM. The ratiometric image indicates that the outer layers of the SFAs are more polar than the inner parts. ( B – D ) Epi-fluorescence images of SFAs prepared in 10 mM Tris, 2 mM CaCl 2 and 100 μM αS stained with ThT and re-suspended in 2 vol% water/n-dodecane emulsion. Under these conditions the SFAs localize in the water phase but remain in contact with the oil phase by exposing their front parts to it. This indicates a more apolar interior of the aggregate. At the front of the SFAs a large part of the interior is exposed to the solvent (shown in A ). Scale bars are 25 μm ( A ) and 100 μm ( D ).
    Figure Legend Snippet: Alignment of αS SFAs at oil/water interface. ( A ) Ratiometric confocal fluorescence image of an αS SFA containing aS140C-MFM. The ratiometric image indicates that the outer layers of the SFAs are more polar than the inner parts. ( B – D ) Epi-fluorescence images of SFAs prepared in 10 mM Tris, 2 mM CaCl 2 and 100 μM αS stained with ThT and re-suspended in 2 vol% water/n-dodecane emulsion. Under these conditions the SFAs localize in the water phase but remain in contact with the oil phase by exposing their front parts to it. This indicates a more apolar interior of the aggregate. At the front of the SFAs a large part of the interior is exposed to the solvent (shown in A ). Scale bars are 25 μm ( A ) and 100 μm ( D ).

    Techniques Used: Fluorescence, Staining

    12) Product Images from "New Insights on the Mechanism of the K+-Independent Activity of Crenarchaeota Pyruvate Kinases"

    Article Title: New Insights on the Mechanism of the K+-Independent Activity of Crenarchaeota Pyruvate Kinases

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0119233

    Double reciprocal plots from the initial velocity data of the Tp PK reaction. The reaction medium consisted of 3 ml of 50 mM Tris-HCl pH 6.0 containing 0.24 mM NADH, 30 mM Mg 2+ free , and 8 μg/ml LDH. The reciprocals of the concentrations of ionized PEP and ADP-Mg complexes are shown in the abscissas of each graph. A . The fixed concentrations of ADP-Mg were 0.084 (■), 0.12 (●), 0.16 (▲), 0.37 (▼), and 0.67 mM (♦). B . The fixed concentrations of PEP 3- were 0.031 (■), 0.14 (●), 0.34 (▲), 0.69(▼), and 1.30 mM (♦). The Mg 2+ free concentration was kept constant at 30 mM. To maintain the ionic strength, (CH 3 ) 4 NCl was added to a final salt concentration of 0.25 M. The reaction was started with the addition of PK. The concentrations of PK were 0.32 and 0.16 μg/ml for the three lowest and two highest substrate concentrations, respectively.
    Figure Legend Snippet: Double reciprocal plots from the initial velocity data of the Tp PK reaction. The reaction medium consisted of 3 ml of 50 mM Tris-HCl pH 6.0 containing 0.24 mM NADH, 30 mM Mg 2+ free , and 8 μg/ml LDH. The reciprocals of the concentrations of ionized PEP and ADP-Mg complexes are shown in the abscissas of each graph. A . The fixed concentrations of ADP-Mg were 0.084 (■), 0.12 (●), 0.16 (▲), 0.37 (▼), and 0.67 mM (♦). B . The fixed concentrations of PEP 3- were 0.031 (■), 0.14 (●), 0.34 (▲), 0.69(▼), and 1.30 mM (♦). The Mg 2+ free concentration was kept constant at 30 mM. To maintain the ionic strength, (CH 3 ) 4 NCl was added to a final salt concentration of 0.25 M. The reaction was started with the addition of PK. The concentrations of PK were 0.32 and 0.16 μg/ml for the three lowest and two highest substrate concentrations, respectively.

    Techniques Used: Concentration Assay

    13) Product Images from "The outer-membrane export signal of Porphyromonas gingivalis type IX secretion system (T9SS) is a conserved C-terminal β-sandwich domain"

    Article Title: The outer-membrane export signal of Porphyromonas gingivalis type IX secretion system (T9SS) is a conserved C-terminal β-sandwich domain

    Journal: Scientific Reports

    doi: 10.1038/srep23123

    Soluble rCTD is a dimer in equilibrium and the CTD cleaved off natively expressed proRgpB spontaneously dimerizes. ( A ) rCTD at 1, 0.5 and 0.1 mg ml −1 was treated with glutaraldehyde and analysed by SDS-PAGE. ( B ) Recombinant CTD (rCTD) (red), proRgpB662iXa (black), and proRgpB662iXa preincubated with fXa (blue) were subjected to size exclusion chromatography on a Superdex 75 10/300 GL column equilibrated with 50 mM Tris, 150 mM NaCl, 2.5 mM CaCl 2 , 0.02% NaN 3 pH 7.5 ( C ) Indicated fractions of resolved proteins were analysed by Western blot using anti-rCTD antibodies to reveal the CTD content in each analysed fraction.
    Figure Legend Snippet: Soluble rCTD is a dimer in equilibrium and the CTD cleaved off natively expressed proRgpB spontaneously dimerizes. ( A ) rCTD at 1, 0.5 and 0.1 mg ml −1 was treated with glutaraldehyde and analysed by SDS-PAGE. ( B ) Recombinant CTD (rCTD) (red), proRgpB662iXa (black), and proRgpB662iXa preincubated with fXa (blue) were subjected to size exclusion chromatography on a Superdex 75 10/300 GL column equilibrated with 50 mM Tris, 150 mM NaCl, 2.5 mM CaCl 2 , 0.02% NaN 3 pH 7.5 ( C ) Indicated fractions of resolved proteins were analysed by Western blot using anti-rCTD antibodies to reveal the CTD content in each analysed fraction.

    Techniques Used: SDS Page, Recombinant, Size-exclusion Chromatography, Western Blot

    14) Product Images from "Covalent modification of phosphatidylethanolamine by 4-hydroxy-2-nonenal increases sodium permeability across phospholipid bilayer membranes"

    Article Title: Covalent modification of phosphatidylethanolamine by 4-hydroxy-2-nonenal increases sodium permeability across phospholipid bilayer membranes

    Journal: Free radical biology & medicine

    doi: 10.1016/j.freeradbiomed.2019.08.027

    Effect of reactive aldehydes (HNE and ONE) on total membrane conductance (Gm). The lipid compositions of bilayer membranes were: DOPC:CL (90:10 mol%), DOPE:DOPC:CL (45:45:10 mol%), and E. coli polar lipid (PE:PG:CL, 71.4:23.4:5.2 mol%). The buffer solution contained 50 mM Na 2 SO 4 , 10 mM MES, and 10 mM TRIS at 32 °C and pH = 7.32. The concentrations of lipids and RAs were 1.2 mg/ml and 0.84 mM, respectively.
    Figure Legend Snippet: Effect of reactive aldehydes (HNE and ONE) on total membrane conductance (Gm). The lipid compositions of bilayer membranes were: DOPC:CL (90:10 mol%), DOPE:DOPC:CL (45:45:10 mol%), and E. coli polar lipid (PE:PG:CL, 71.4:23.4:5.2 mol%). The buffer solution contained 50 mM Na 2 SO 4 , 10 mM MES, and 10 mM TRIS at 32 °C and pH = 7.32. The concentrations of lipids and RAs were 1.2 mg/ml and 0.84 mM, respectively.

    Techniques Used:

    Influence of HNE-DOPE adducts on Na + and H + translocation. A. Representative current-voltage measurements in the presence (grey triangles) and absence (white dots) of a pH gradient of 0.4. The voltage shift is the difference in the x-axis intersection values of both measurements. The membranes were composed of DOPE:DOPC:CL (45:45:10 mol%). The buffer solution contained 50 mM Na 2 SO 4 , 10 mM MES, 10 mM TRIS, and 0.6 mM EGTA at 32°C and pH=7.32. B. Influence of the ionic strength of the buffer solution on total membrane conductance (Gm) in the presence of HNE or ONE. The membrane were composed of DOPE:DOPC (50:50 mol%). Besides NaCl, the buffer solution also contained 10 mM MES and 10 mM TRIS at 32°C and pH 7.32. The concentrations of lipids and RAs were 1.2 mg/ml and 0.84 mM, respectively.
    Figure Legend Snippet: Influence of HNE-DOPE adducts on Na + and H + translocation. A. Representative current-voltage measurements in the presence (grey triangles) and absence (white dots) of a pH gradient of 0.4. The voltage shift is the difference in the x-axis intersection values of both measurements. The membranes were composed of DOPE:DOPC:CL (45:45:10 mol%). The buffer solution contained 50 mM Na 2 SO 4 , 10 mM MES, 10 mM TRIS, and 0.6 mM EGTA at 32°C and pH=7.32. B. Influence of the ionic strength of the buffer solution on total membrane conductance (Gm) in the presence of HNE or ONE. The membrane were composed of DOPE:DOPC (50:50 mol%). Besides NaCl, the buffer solution also contained 10 mM MES and 10 mM TRIS at 32°C and pH 7.32. The concentrations of lipids and RAs were 1.2 mg/ml and 0.84 mM, respectively.

    Techniques Used: Translocation Assay

    15) Product Images from "Molecular Model for the Solubilization of Membranes into Nanodisks by Styrene Maleic Acid Copolymers"

    Article Title: Molecular Model for the Solubilization of Membranes into Nanodisks by Styrene Maleic Acid Copolymers

    Journal: Biophysical Journal

    doi: 10.1016/j.bpj.2014.11.3464

    Effect of electrostatic interactions on membrane insertion and solubilization by SMA copolymers. ( A ) Surface pressure increase in time upon the addition of excess SMA to monolayers of di-14:0 PC ( red ), di-14:0 PC/PG (4:1) ( orange ), and di-14:0 PG ( blue ) in 50 mM Tris-HCl pH 8.0 with 150 mM NaCl ( solid ) or without NaCl ( dashed ). ( B ) Surface pressure increase as function of initial surface pressure for di-14:0 PC and di-14:0 PG lipid monolayers in 50 mM Tris-HCl pH 8.0 with 150 mM NaCl as subphase. ( Solid lines ) Linear fit with the maximum insertion pressure extrapolated to be ∼48 mN/m. Error bars are standard errors based on at least two independent measurements. ( C ) Normalized absorbance values at 350 nm of lipid vesicle dispersions after 10 min of incubation with the SMA copolymer at different temperatures. The diameter of the vesicles was 400 nm and a 3:1 (w/w) SMA/lipid was used. ( Solid lines ) Data recorded in Tris-HCl pH 8.0 buffer with 150 mM NaCl; ( dashed lines ) without NaCl. To see this figure in color, go online.
    Figure Legend Snippet: Effect of electrostatic interactions on membrane insertion and solubilization by SMA copolymers. ( A ) Surface pressure increase in time upon the addition of excess SMA to monolayers of di-14:0 PC ( red ), di-14:0 PC/PG (4:1) ( orange ), and di-14:0 PG ( blue ) in 50 mM Tris-HCl pH 8.0 with 150 mM NaCl ( solid ) or without NaCl ( dashed ). ( B ) Surface pressure increase as function of initial surface pressure for di-14:0 PC and di-14:0 PG lipid monolayers in 50 mM Tris-HCl pH 8.0 with 150 mM NaCl as subphase. ( Solid lines ) Linear fit with the maximum insertion pressure extrapolated to be ∼48 mN/m. Error bars are standard errors based on at least two independent measurements. ( C ) Normalized absorbance values at 350 nm of lipid vesicle dispersions after 10 min of incubation with the SMA copolymer at different temperatures. The diameter of the vesicles was 400 nm and a 3:1 (w/w) SMA/lipid was used. ( Solid lines ) Data recorded in Tris-HCl pH 8.0 buffer with 150 mM NaCl; ( dashed lines ) without NaCl. To see this figure in color, go online.

    Techniques Used: Incubation

    16) Product Images from "The Major Histocompatibility Complex-related Fc Receptor for IgG (FcRn) Binds Albumin and Prolongs Its Lifespan"

    Article Title: The Major Histocompatibility Complex-related Fc Receptor for IgG (FcRn) Binds Albumin and Prolongs Its Lifespan

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20021829

    Detergent inhibits shFcRn albumin interaction. Sepharose (S)-HSA, S-IgG and S-Tris (A–C) were incubated with shFcRn in a pH 6.0 buffer containing indicated concentrations of OG as shown. Bound shFcRn was eluted and quantified by immunoblotting with anti-FcRn antibody. The positions of molecular weight markers (M, in kD) are shown. Lane 1 in each case shows the eluate from Sepharose-ligand in the absence of shFcRn. Lane 2 in all the gels contained 10 μg shFcRn, the amount added to every adsorbent sample. The shFcRn bands were quantified and plotted in panel D vs. OG concentration. Similar results were obtained in another experiment.
    Figure Legend Snippet: Detergent inhibits shFcRn albumin interaction. Sepharose (S)-HSA, S-IgG and S-Tris (A–C) were incubated with shFcRn in a pH 6.0 buffer containing indicated concentrations of OG as shown. Bound shFcRn was eluted and quantified by immunoblotting with anti-FcRn antibody. The positions of molecular weight markers (M, in kD) are shown. Lane 1 in each case shows the eluate from Sepharose-ligand in the absence of shFcRn. Lane 2 in all the gels contained 10 μg shFcRn, the amount added to every adsorbent sample. The shFcRn bands were quantified and plotted in panel D vs. OG concentration. Similar results were obtained in another experiment.

    Techniques Used: Incubation, Molecular Weight, Concentration Assay

    pH-dependent binding of FcRn to immobilized albumin. Sepharose (S)-HSA, S-IgG, S-Tris, S-fish gelatin (see Materials and Methods), or S-RSA were incubated with shFcRn or srFcRn at varying pH as shown. Bound soluble FcRn were eluted and were quantified by immunoblotting with anti-FcRn antibody. Immunoblots of binding of shFcRn to S-HSA, S-IgG, and S-Tris at pH values indicated are shown in A, B, and C, respectively. Immunoblot of binding of srFcRn to S-RSA is shown in E. The positions of molecular weight markers (M, in kD) are shown. Lane 1 in all the gels contained 15 μg soluble FcRn, the amount added to every adsorbent sample. Lane 2 in each case shows the eluate from Sepharose-ligand in the absence of FcRn. The shFcRn bands were quantified and are plotted in panel D vs. pH. Three distinct experiments have given equivalent results. srFcRn band densities are plotted in panel F vs. pH. A second experiment with srFcRn gave equivalent results. G and H show binding of HFE to S-HSA and S-myoglobin. Lanes 1 and 2 show amounts of shFcRn and HFE added (15 μg) to adsorbent samples. Bound protein was eluted and quantified by blotting with anti-b2m antibody.
    Figure Legend Snippet: pH-dependent binding of FcRn to immobilized albumin. Sepharose (S)-HSA, S-IgG, S-Tris, S-fish gelatin (see Materials and Methods), or S-RSA were incubated with shFcRn or srFcRn at varying pH as shown. Bound soluble FcRn were eluted and were quantified by immunoblotting with anti-FcRn antibody. Immunoblots of binding of shFcRn to S-HSA, S-IgG, and S-Tris at pH values indicated are shown in A, B, and C, respectively. Immunoblot of binding of srFcRn to S-RSA is shown in E. The positions of molecular weight markers (M, in kD) are shown. Lane 1 in all the gels contained 15 μg soluble FcRn, the amount added to every adsorbent sample. Lane 2 in each case shows the eluate from Sepharose-ligand in the absence of FcRn. The shFcRn bands were quantified and are plotted in panel D vs. pH. Three distinct experiments have given equivalent results. srFcRn band densities are plotted in panel F vs. pH. A second experiment with srFcRn gave equivalent results. G and H show binding of HFE to S-HSA and S-myoglobin. Lanes 1 and 2 show amounts of shFcRn and HFE added (15 μg) to adsorbent samples. Bound protein was eluted and quantified by blotting with anti-b2m antibody.

    Techniques Used: Binding Assay, Fluorescence In Situ Hybridization, Incubation, Western Blot, Molecular Weight

    17) Product Images from "SARS-CoV-2 nucleocapsid protein undergoes liquid-liquid phase separation stimulated by RNA and partitions into phases of human ribonucleoproteins"

    Article Title: SARS-CoV-2 nucleocapsid protein undergoes liquid-liquid phase separation stimulated by RNA and partitions into phases of human ribonucleoproteins

    Journal: bioRxiv

    doi: 10.1101/2020.06.09.141101

    SARS-CoV-2 nucleocapsid protein undergoes LLPS at physiological conditions. A) Phase separation over time as monitored by turbidity of 50 μM MBP-N after addition of TEV protease in varying pH conditions. B-C) DIC micrographs of 50 μM MBP-N in 50 mM Tris 183 mM NaCl pH 7.4 or 20 mM MES 183 mM NaCl pH 5.5 with and without TEV protease (to cleave MBP from N) or 0.3 mg/mL desalted total torula yeast RNA. Scale bars represent 50 μm.
    Figure Legend Snippet: SARS-CoV-2 nucleocapsid protein undergoes LLPS at physiological conditions. A) Phase separation over time as monitored by turbidity of 50 μM MBP-N after addition of TEV protease in varying pH conditions. B-C) DIC micrographs of 50 μM MBP-N in 50 mM Tris 183 mM NaCl pH 7.4 or 20 mM MES 183 mM NaCl pH 5.5 with and without TEV protease (to cleave MBP from N) or 0.3 mg/mL desalted total torula yeast RNA. Scale bars represent 50 μm.

    Techniques Used:

    SARS-CoV-2 N LLPS is modulated by salt and RNA. A-B) Phase separation over time as monitored by turbidity of 50 μM MBP-N in 50 mM Tris pH 7.4 after addition of TEV protease with varying torula yeast RNA (at 100 mM sodium chloride) or varying sodium chloride concentrations. C-D) DIC micrographs of 50 μM MBP-N in 50 mM Tris NaCl pH 7.4 with varying torula yeast RNA or sodium chloride concentrations with and without TEV protease (to cleave MBP from N). Scale bars represent 50 μm.
    Figure Legend Snippet: SARS-CoV-2 N LLPS is modulated by salt and RNA. A-B) Phase separation over time as monitored by turbidity of 50 μM MBP-N in 50 mM Tris pH 7.4 after addition of TEV protease with varying torula yeast RNA (at 100 mM sodium chloride) or varying sodium chloride concentrations. C-D) DIC micrographs of 50 μM MBP-N in 50 mM Tris NaCl pH 7.4 with varying torula yeast RNA or sodium chloride concentrations with and without TEV protease (to cleave MBP from N). Scale bars represent 50 μm.

    Techniques Used:

    18) Product Images from "Isolation and characterization of a novel phage lysin active against Paenibacillus larvae, a honeybee pathogen"

    Article Title: Isolation and characterization of a novel phage lysin active against Paenibacillus larvae, a honeybee pathogen

    Journal: Bacteriophage

    doi: 10.1080/21597081.2015.1080787

    SDS-PAGE of lysin purification after AEC, IMAC, thrombin digest, and more AEC. The 10% acrylamide gel was run at 60V for 30 mins followed by 150V for 60 mins in buffer containing 50 mM Tris, 50 mM MOPS, 0.1% SDS, and 1 mM EDTA, pH 6.8. The lanes from left to right: (1) 10-250 kDa Protein Ladder (New England Biolabs), (2) 210 mM NaCl eluate from second AEC after thrombin digest, (3) 250 mM imidazole eluate from IMAC, (4) 25 mM imidazole eluate from IMAC, (5) 200 mM NaCl eluate from first AEC, (6) crude B-PER lysate of IPTG-induced E. coli , (7) 10-250 kDa Protein Ladder (New England Biolabs).
    Figure Legend Snippet: SDS-PAGE of lysin purification after AEC, IMAC, thrombin digest, and more AEC. The 10% acrylamide gel was run at 60V for 30 mins followed by 150V for 60 mins in buffer containing 50 mM Tris, 50 mM MOPS, 0.1% SDS, and 1 mM EDTA, pH 6.8. The lanes from left to right: (1) 10-250 kDa Protein Ladder (New England Biolabs), (2) 210 mM NaCl eluate from second AEC after thrombin digest, (3) 250 mM imidazole eluate from IMAC, (4) 25 mM imidazole eluate from IMAC, (5) 200 mM NaCl eluate from first AEC, (6) crude B-PER lysate of IPTG-induced E. coli , (7) 10-250 kDa Protein Ladder (New England Biolabs).

    Techniques Used: SDS Page, Purification, Acrylamide Gel Assay

    19) Product Images from "The outer-membrane export signal of Porphyromonas gingivalis type IX secretion system (T9SS) is a conserved C-terminal β-sandwich domain"

    Article Title: The outer-membrane export signal of Porphyromonas gingivalis type IX secretion system (T9SS) is a conserved C-terminal β-sandwich domain

    Journal: Scientific Reports

    doi: 10.1038/srep23123

    Soluble rCTD is a dimer in equilibrium and the CTD cleaved off natively expressed proRgpB spontaneously dimerizes. ( A ) rCTD at 1, 0.5 and 0.1 mg ml −1 was treated with glutaraldehyde and analysed by SDS-PAGE. ( B ) Recombinant CTD (rCTD) (red), proRgpB662iXa (black), and proRgpB662iXa preincubated with fXa (blue) were subjected to size exclusion chromatography on a Superdex 75 10/300 GL column equilibrated with 50 mM Tris, 150 mM NaCl, 2.5 mM CaCl 2 , 0.02% NaN 3 pH 7.5 ( C ) Indicated fractions of resolved proteins were analysed by Western blot using anti-rCTD antibodies to reveal the CTD content in each analysed fraction.
    Figure Legend Snippet: Soluble rCTD is a dimer in equilibrium and the CTD cleaved off natively expressed proRgpB spontaneously dimerizes. ( A ) rCTD at 1, 0.5 and 0.1 mg ml −1 was treated with glutaraldehyde and analysed by SDS-PAGE. ( B ) Recombinant CTD (rCTD) (red), proRgpB662iXa (black), and proRgpB662iXa preincubated with fXa (blue) were subjected to size exclusion chromatography on a Superdex 75 10/300 GL column equilibrated with 50 mM Tris, 150 mM NaCl, 2.5 mM CaCl 2 , 0.02% NaN 3 pH 7.5 ( C ) Indicated fractions of resolved proteins were analysed by Western blot using anti-rCTD antibodies to reveal the CTD content in each analysed fraction.

    Techniques Used: SDS Page, Recombinant, Size-exclusion Chromatography, Western Blot

    20) Product Images from "Direct Observation of the Interconversion of Normal and Toxic Forms of ?-Synuclein"

    Article Title: Direct Observation of the Interconversion of Normal and Toxic Forms of ?-Synuclein

    Journal: Cell

    doi: 10.1016/j.cell.2012.03.037

    Characterization of Labeled A90C αS at Bulk Conditions, Related to Figure 1 (A) αS aminoacidic sequence, highlighting the three main regions: N-terminal (residues 1-60), NAC (61-95) and C-terminal (95-140); and the five regions proposed to form the strands of the beta-sheet sandwich core of the fibrillar structure ( Vilar et al., 2008 ): strand β1 comprising residues 37-43; strand β2, 52-59; strand β3, 62-66; strand β4, 68-77 and strand β5, 90-95. The fluorescence dyes were covalently attached to position 90 in the sequence by cysteine chemistry. (B) The fold of αS fibrillar structure proposed by Vilar et al. ( Vilar et al., 2008 ) is shown. The fluorescence dyes would be positioned parallel at the periphery of the fibril core according to this model. (C) Dynamic light scattering derived size distribution of unincubated AF488 A90C αS (red line) compared with unlabeled WT protein (black line). Representative size distribution of 30 μM protein concentration in Tris 25 mM, pH 7.4, 0.1 M NaCl at 25°C measured on the Zetasizer Nano ZS instrument at 633nm. The distribution represents the average of 4 measurements and the intensity was normalized and weighted by the particle size. The influence of AF647 could not be measured with our DLS instrument due to the direct absorption of the laser light (633 nm) by the fluorophore, although very similar results are expected. (D) The kinetics of fibril formation for the unlabeled protein was independently analyzed by the addition of Thioflavin T (ThT) to the reaction sample at different incubation times (open circles: the signal was normalized and 1-(normalized signal) was plotted to compared with the kinetics of the depletion of the monomeric protein analyzed by SEC). The decrease in monomeric protein during incubation was also estimated by quantitative SEC analysis after centrifuging the samples to remove the insoluble material (closed circles) or after ultracentrifugation to remove big soluble oligomers, but no differences were observed. The lag time and apparent kinetic rate were obtained. Error bars represent SEM. (E) For the case of labeled protein, analysis using ThT cannot be applied, since the fluorescence increase of ThT molecules upon binding to the amyloid fibrils is significantly reduced, probably due to fluorescence quenching and/or FRET between ThT and the Alexa fluorophores. For this reason, the kinetics of fibril formation was followed by quantitative SEC (closed circles; see Figure S1 G) and SDS-PAGE gel (open circles), where the soluble protein material was quantified by the spectroscopic properties of the Alexa fluorophores. The aggregation of mixed AF488 and AF647-labeled protein is shown as blue circles, and the aggregation corresponding to AF488-labeled protein incubated alone is shown in red. Error bars represent SEM. (F) TEM images show that the morphology of the labeled fibrils formed (image on the right) is very similar to those formed with unmodified protein (image on the left). Amorphous aggregates were not observed in any case. (G) Oligomers were detected and analyzed by quantitative SEC as a function of incubation time. Three peaks were observed in the chromatogram: a peak eluting at 7 ml, which corresponds to the column void volume, and then to oligomeric species, a large peak eluting at 9.3 ml which corresponds to the monomeric protein, and a third peak at eluting volumes bigger than 15 ml, corresponding to some fragments of the protein generated upon incubation. The concentration of both monomeric and oligomeric fractions of the protein at the different incubation times recorded were estimated from the area of the peaks, taking into account the known initial concentration of protein and that at time zero, all the protein remains monomeric.
    Figure Legend Snippet: Characterization of Labeled A90C αS at Bulk Conditions, Related to Figure 1 (A) αS aminoacidic sequence, highlighting the three main regions: N-terminal (residues 1-60), NAC (61-95) and C-terminal (95-140); and the five regions proposed to form the strands of the beta-sheet sandwich core of the fibrillar structure ( Vilar et al., 2008 ): strand β1 comprising residues 37-43; strand β2, 52-59; strand β3, 62-66; strand β4, 68-77 and strand β5, 90-95. The fluorescence dyes were covalently attached to position 90 in the sequence by cysteine chemistry. (B) The fold of αS fibrillar structure proposed by Vilar et al. ( Vilar et al., 2008 ) is shown. The fluorescence dyes would be positioned parallel at the periphery of the fibril core according to this model. (C) Dynamic light scattering derived size distribution of unincubated AF488 A90C αS (red line) compared with unlabeled WT protein (black line). Representative size distribution of 30 μM protein concentration in Tris 25 mM, pH 7.4, 0.1 M NaCl at 25°C measured on the Zetasizer Nano ZS instrument at 633nm. The distribution represents the average of 4 measurements and the intensity was normalized and weighted by the particle size. The influence of AF647 could not be measured with our DLS instrument due to the direct absorption of the laser light (633 nm) by the fluorophore, although very similar results are expected. (D) The kinetics of fibril formation for the unlabeled protein was independently analyzed by the addition of Thioflavin T (ThT) to the reaction sample at different incubation times (open circles: the signal was normalized and 1-(normalized signal) was plotted to compared with the kinetics of the depletion of the monomeric protein analyzed by SEC). The decrease in monomeric protein during incubation was also estimated by quantitative SEC analysis after centrifuging the samples to remove the insoluble material (closed circles) or after ultracentrifugation to remove big soluble oligomers, but no differences were observed. The lag time and apparent kinetic rate were obtained. Error bars represent SEM. (E) For the case of labeled protein, analysis using ThT cannot be applied, since the fluorescence increase of ThT molecules upon binding to the amyloid fibrils is significantly reduced, probably due to fluorescence quenching and/or FRET between ThT and the Alexa fluorophores. For this reason, the kinetics of fibril formation was followed by quantitative SEC (closed circles; see Figure S1 G) and SDS-PAGE gel (open circles), where the soluble protein material was quantified by the spectroscopic properties of the Alexa fluorophores. The aggregation of mixed AF488 and AF647-labeled protein is shown as blue circles, and the aggregation corresponding to AF488-labeled protein incubated alone is shown in red. Error bars represent SEM. (F) TEM images show that the morphology of the labeled fibrils formed (image on the right) is very similar to those formed with unmodified protein (image on the left). Amorphous aggregates were not observed in any case. (G) Oligomers were detected and analyzed by quantitative SEC as a function of incubation time. Three peaks were observed in the chromatogram: a peak eluting at 7 ml, which corresponds to the column void volume, and then to oligomeric species, a large peak eluting at 9.3 ml which corresponds to the monomeric protein, and a third peak at eluting volumes bigger than 15 ml, corresponding to some fragments of the protein generated upon incubation. The concentration of both monomeric and oligomeric fractions of the protein at the different incubation times recorded were estimated from the area of the peaks, taking into account the known initial concentration of protein and that at time zero, all the protein remains monomeric.

    Techniques Used: Labeling, Sequencing, Fluorescence, Derivative Assay, Protein Concentration, Incubation, Size-exclusion Chromatography, Binding Assay, SDS Page, Transmission Electron Microscopy, Generated, Concentration Assay

    21) Product Images from "Engineering the “Missing Link” in Biosynthetic (−)-Menthol Production: Bacterial Isopulegone Isomerase"

    Article Title: Engineering the “Missing Link” in Biosynthetic (−)-Menthol Production: Bacterial Isopulegone Isomerase

    Journal: ACS Catalysis

    doi: 10.1021/acscatal.7b04115

    Location of key active site mutations implicated in improving KSI activity toward 3 . (A) Residues located in the equilenin-binding region of wild type KSI from P. putida The residues and equilenin are shown as atom colored sticks with yellow and green carbons, respectively. Interactions are shown as red dotted lines. The backbone is shown as a gray cartoon. (B) Comparative steady-state activity of wild-type and variant KSI enzymes. Reaction mixtures (100 μL) were composed of 50 mM Tris pH 7.0 containing 1 mM 3 . The absorbance was monitored at 260 nm for 1 h at 20 °C. Inset: location of the variant residues in KSI V881/L99V/D103S. The backbone and mutations are shown as gray ribbons and balls, respectively (blue balls for V101).
    Figure Legend Snippet: Location of key active site mutations implicated in improving KSI activity toward 3 . (A) Residues located in the equilenin-binding region of wild type KSI from P. putida The residues and equilenin are shown as atom colored sticks with yellow and green carbons, respectively. Interactions are shown as red dotted lines. The backbone is shown as a gray cartoon. (B) Comparative steady-state activity of wild-type and variant KSI enzymes. Reaction mixtures (100 μL) were composed of 50 mM Tris pH 7.0 containing 1 mM 3 . The absorbance was monitored at 260 nm for 1 h at 20 °C. Inset: location of the variant residues in KSI V881/L99V/D103S. The backbone and mutations are shown as gray ribbons and balls, respectively (blue balls for V101).

    Techniques Used: Activity Assay, Binding Assay, Variant Assay

    22) Product Images from "Effect of Fengycin, a Lipopeptide Produced by Bacillus subtilis, on Model Biomembranes"

    Article Title: Effect of Fengycin, a Lipopeptide Produced by Bacillus subtilis, on Model Biomembranes

    Journal:

    doi: 10.1529/biophysj.107.114090

    Second heating scan of fengycin and/or DPPC vesicles in a 10 mM Tris, 150 mM NaCl buffer at pH 7.4. ( a ) DPPC vesicles 1 mM; ( b ) DPPC/fengycin (300:1), [fengycin] = 3.3 μ M; ( c ) DPPC/fengycin (37.5:1), [fengycin] = 25.8 μ
    Figure Legend Snippet: Second heating scan of fengycin and/or DPPC vesicles in a 10 mM Tris, 150 mM NaCl buffer at pH 7.4. ( a ) DPPC vesicles 1 mM; ( b ) DPPC/fengycin (300:1), [fengycin] = 3.3 μ M; ( c ) DPPC/fengycin (37.5:1), [fengycin] = 25.8 μ

    Techniques Used:

    Surface pressure increase after injection of fengycin under a DPPC monolayer previously compressed at a defined initial surface pressure. Subphase is a 10 mM Tris, 150 mM NaCl at pH 7.4. Temperature is 30°C. Fengycin is injected into the subphase
    Figure Legend Snippet: Surface pressure increase after injection of fengycin under a DPPC monolayer previously compressed at a defined initial surface pressure. Subphase is a 10 mM Tris, 150 mM NaCl at pH 7.4. Temperature is 30°C. Fengycin is injected into the subphase

    Techniques Used: Injection

    23) Product Images from "Novel Methylselenoesters as Antiproliferative Agents"

    Article Title: Novel Methylselenoesters as Antiproliferative Agents

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    doi: 10.3390/molecules22081288

    Compounds 5 and 15 are substrates for thioredoxin reductase but not for the glutathione-glutaredoxin system. ( A ) NADPH consumption indicating reduction of compounds 5 , 15 or MSA as control by thioredoxin reductase. The reaction mixture contained 100 nM TrxR1, 227 μM NADPH and the corresponding amount of compound in TE buffer (20 mM Tris, 2 mM EDTA pH = 8); ( B ) NADPH consumption indicating reduction of MSA by glutathione in the presence or absence of glutaredoxin. The reaction mixture contained the corresponding amount of compound, 0.1 M Tris, 2 mM EDTA pH = 8, 0.1 mg/mL BSA, 1 mM GSH, 200 μM NADPH, 0.008 OD/mL yeast GR and 1 μM hGrx1 when required. Only results for MSA are shown, as compounds 5 and 15 were not reduced.
    Figure Legend Snippet: Compounds 5 and 15 are substrates for thioredoxin reductase but not for the glutathione-glutaredoxin system. ( A ) NADPH consumption indicating reduction of compounds 5 , 15 or MSA as control by thioredoxin reductase. The reaction mixture contained 100 nM TrxR1, 227 μM NADPH and the corresponding amount of compound in TE buffer (20 mM Tris, 2 mM EDTA pH = 8); ( B ) NADPH consumption indicating reduction of MSA by glutathione in the presence or absence of glutaredoxin. The reaction mixture contained the corresponding amount of compound, 0.1 M Tris, 2 mM EDTA pH = 8, 0.1 mg/mL BSA, 1 mM GSH, 200 μM NADPH, 0.008 OD/mL yeast GR and 1 μM hGrx1 when required. Only results for MSA are shown, as compounds 5 and 15 were not reduced.

    Techniques Used:

    24) Product Images from "Mycobacterium tuberculosis Hip1 Modulates Macrophage Responses through Proteolysis of GroEL2"

    Article Title: Mycobacterium tuberculosis Hip1 Modulates Macrophage Responses through Proteolysis of GroEL2

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1004132

    Analysis of the enzymatic activity of Hip1. (A) Hip1 esterase activity with p- nitrophenylbutyrate. P- nitrophenylbutyrate was incubated alone for a negative control reaction (-control). PreScission protease was used in a positive control reaction (+ control). (B) Azocasein proteolysis assay showing that Hip1 is a protease. Azocasein was incubated alone (- control), with the protease subtilisin (+control), with Hip1 (0.05 mg/ml), or Hip1(S228A) (0.05 mg/ml) in 25 mM Tris pH 7.4, 150 mM NaCl. The enzyme activities are expressed as units of enzyme/mg protein (one enzyme unit is the quantity of enzyme required to increase absorbance by 0.01 units at 440 nm). (C) Endpoint assay showing proteolytic activity of Hip1. Hip1 (7.5 µM) was incubated with each peptide substrate (1.5 mM) or alone (-control) in 50 mM Tris pH 8.0 for 18 hr at 25°C. Elastase was used as a positive control (+ control). Hydrolysis of the peptide substrates was detected by monitoring an increase in absorbance at 410 nm. (D) Inhibition of Hip1 with various classes of protease inhibitors. Hip1 (4 µM) was pre-incubated with inhibitor for 30 min in 50 mM Tris, pH 8.0 at 25°C. Then, protease activity was measured by the addition of 1.5 mM Ala-Pro-Ala- p Na. The specific activity of Hip1 against Ala-Pro-Ala- p Na was defined as 100% (no inhibitor). Data are shown as one representative experiment from three independent experiments.
    Figure Legend Snippet: Analysis of the enzymatic activity of Hip1. (A) Hip1 esterase activity with p- nitrophenylbutyrate. P- nitrophenylbutyrate was incubated alone for a negative control reaction (-control). PreScission protease was used in a positive control reaction (+ control). (B) Azocasein proteolysis assay showing that Hip1 is a protease. Azocasein was incubated alone (- control), with the protease subtilisin (+control), with Hip1 (0.05 mg/ml), or Hip1(S228A) (0.05 mg/ml) in 25 mM Tris pH 7.4, 150 mM NaCl. The enzyme activities are expressed as units of enzyme/mg protein (one enzyme unit is the quantity of enzyme required to increase absorbance by 0.01 units at 440 nm). (C) Endpoint assay showing proteolytic activity of Hip1. Hip1 (7.5 µM) was incubated with each peptide substrate (1.5 mM) or alone (-control) in 50 mM Tris pH 8.0 for 18 hr at 25°C. Elastase was used as a positive control (+ control). Hydrolysis of the peptide substrates was detected by monitoring an increase in absorbance at 410 nm. (D) Inhibition of Hip1 with various classes of protease inhibitors. Hip1 (4 µM) was pre-incubated with inhibitor for 30 min in 50 mM Tris, pH 8.0 at 25°C. Then, protease activity was measured by the addition of 1.5 mM Ala-Pro-Ala- p Na. The specific activity of Hip1 against Ala-Pro-Ala- p Na was defined as 100% (no inhibitor). Data are shown as one representative experiment from three independent experiments.

    Techniques Used: Activity Assay, Incubation, Negative Control, Positive Control, Proteolysis Assay, End Point Assay, Inhibition

    25) Product Images from "Elucidation of IP6 and Heparin Interaction Sites and Conformational Changes in Arrestin-1 by Solution NMR †"

    Article Title: Elucidation of IP6 and Heparin Interaction Sites and Conformational Changes in Arrestin-1 by Solution NMR †

    Journal: Biochemistry

    doi: 10.1021/bi101596g

    2D 1 H- 15 N TROSY spectrum of 0.2mM selectively 15 N-isoleucine-labeled arrestin-1 in 25 mM Bis-Tris, 150mM NaCl and 5mM mercaptoethanol, pH=6.5 at 308 K using a Bruker Avance 800MHz spectrometer. 11 out of 20 isoleucine residue peaks were assigned, as labeled
    Figure Legend Snippet: 2D 1 H- 15 N TROSY spectrum of 0.2mM selectively 15 N-isoleucine-labeled arrestin-1 in 25 mM Bis-Tris, 150mM NaCl and 5mM mercaptoethanol, pH=6.5 at 308 K using a Bruker Avance 800MHz spectrometer. 11 out of 20 isoleucine residue peaks were assigned, as labeled

    Techniques Used: Labeling

    2-D 1 H- 15 N TROSY spectrum of 0.2mM U- 2 H, 15 N-arrestin-1 in 25 mM Bis-Tris, 150 mM NaCl, 5 mM mercaptoethanol, pH=6.5 acquired at 308 K using a Bruker Avance 800MHz spectrometer. 152 assigned residues are labeled.
    Figure Legend Snippet: 2-D 1 H- 15 N TROSY spectrum of 0.2mM U- 2 H, 15 N-arrestin-1 in 25 mM Bis-Tris, 150 mM NaCl, 5 mM mercaptoethanol, pH=6.5 acquired at 308 K using a Bruker Avance 800MHz spectrometer. 152 assigned residues are labeled.

    Techniques Used: Labeling

    26) Product Images from "Influence of specific HSP70 domains on fibril formation of the yeast prion protein Ure2"

    Article Title: Influence of specific HSP70 domains on fibril formation of the yeast prion protein Ure2

    Journal: Philosophical Transactions of the Royal Society B: Biological Sciences

    doi: 10.1098/rstb.2011.0410

    Ssa1p inhibits fibril formation of Ure2p in a concentration-dependent manner. Incubation was in a Perkin-Elmer EnSpire multimode plate reader at 30°C in Tris buffer pH 7.5. Fibril formation was monitored by in situ measurement of ThT fluorescence. The error bars represent the standard error of the mean. Data were normalized. Other details are as described in §2. Ure2p (30 μM) in the absence or presence of different molar ratios of ( a ) WT Ssa1p or ( b ) Δ A Ssa1p, as indicated. The data are fitted (solid lines) globally with an analytical model of breakable fibril assembly (see §2); this constrained fit, where only the rate of elongation is affected by the chaperone, is able to describe the entire dataset. The insets show the variation in the relative elongation rate with chaperone concentration, as determined by the fitted rate parameters.
    Figure Legend Snippet: Ssa1p inhibits fibril formation of Ure2p in a concentration-dependent manner. Incubation was in a Perkin-Elmer EnSpire multimode plate reader at 30°C in Tris buffer pH 7.5. Fibril formation was monitored by in situ measurement of ThT fluorescence. The error bars represent the standard error of the mean. Data were normalized. Other details are as described in §2. Ure2p (30 μM) in the absence or presence of different molar ratios of ( a ) WT Ssa1p or ( b ) Δ A Ssa1p, as indicated. The data are fitted (solid lines) globally with an analytical model of breakable fibril assembly (see §2); this constrained fit, where only the rate of elongation is affected by the chaperone, is able to describe the entire dataset. The insets show the variation in the relative elongation rate with chaperone concentration, as determined by the fitted rate parameters.

    Techniques Used: Concentration Assay, Incubation, In Situ, Fluorescence

    27) Product Images from "Calretinin and calbindin D28k have different domain organizations"

    Article Title: Calretinin and calbindin D28k have different domain organizations

    Journal: Protein Science : A Publication of the Protein Society

    doi:

    NMR analysis of CR and CR fragments in 50 mM Tris, 10 mM CaCl 2 , 20 mM NaCl, pH 7.7 buffer. ( A ) 1 H, 15 N HSQC spectra of 50 μM 15 N-labeled CR III–VI in the absence (black) and presence (red) of an equivalent concentration of unlabeled CR I–II. ( B ) Overlaid 1 H, 15 N HSQC spectra of CR I–II (black) and CR III–VI (red). ( C ) 1 H, 15 N HSQC spectrum of CR. The pattern of peaks from CR resembles the summed spectra of CR I–II and CR III–VI, particularly in well dispersed areas of the spectra. All spectra in panels B and C were obtained on separate samples.
    Figure Legend Snippet: NMR analysis of CR and CR fragments in 50 mM Tris, 10 mM CaCl 2 , 20 mM NaCl, pH 7.7 buffer. ( A ) 1 H, 15 N HSQC spectra of 50 μM 15 N-labeled CR III–VI in the absence (black) and presence (red) of an equivalent concentration of unlabeled CR I–II. ( B ) Overlaid 1 H, 15 N HSQC spectra of CR I–II (black) and CR III–VI (red). ( C ) 1 H, 15 N HSQC spectrum of CR. The pattern of peaks from CR resembles the summed spectra of CR I–II and CR III–VI, particularly in well dispersed areas of the spectra. All spectra in panels B and C were obtained on separate samples.

    Techniques Used: Nuclear Magnetic Resonance, Labeling, Concentration Assay

    28) Product Images from "HIV-1 Envelope Resistance to Proteasomal Cleavage: Implications for Vaccine Induced Immune Responses"

    Article Title: HIV-1 Envelope Resistance to Proteasomal Cleavage: Implications for Vaccine Induced Immune Responses

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0042579

    CAT are able to cleave HIV-1 Env-A244 and CAT degradation products are susceptible to proteasomal degradation. HIV-1 Env-A244 (panels A and B, lanes 0) was treated with CAT for 90 min or 16 hrs and then analyzed on a 4–20% gradient Tris-glycine polyacrylamide gel. The 90 min (lanes 90) and 16 hrs (lanes 0/N) degradation pattern with the various CAT are shown in panels A and B. Env-A244 was treated with the individual CAT for 90 min followed by proteasomes in the presence (panels C and D, lanes PI) or absence of epoxomicin (lanes P). The molecular weight markers are shown in lane M in all panels.
    Figure Legend Snippet: CAT are able to cleave HIV-1 Env-A244 and CAT degradation products are susceptible to proteasomal degradation. HIV-1 Env-A244 (panels A and B, lanes 0) was treated with CAT for 90 min or 16 hrs and then analyzed on a 4–20% gradient Tris-glycine polyacrylamide gel. The 90 min (lanes 90) and 16 hrs (lanes 0/N) degradation pattern with the various CAT are shown in panels A and B. Env-A244 was treated with the individual CAT for 90 min followed by proteasomes in the presence (panels C and D, lanes PI) or absence of epoxomicin (lanes P). The molecular weight markers are shown in lane M in all panels.

    Techniques Used: Molecular Weight

    Proteasomes are unable to cleave HIV-1 Env proteins. Env-A244 (CRF01_AE) derived from CHO cells and gp140 (clade B) derived from H9 cells (panel A, lanes C) were treated with purified proteasomes isolated from activated CD4 + T-cells in the absence (lanes, P) or presence of epoxomicin, an irreversible proteasome inhibitor (lanes PI). Proteasomes were unable to cleave the Env proteins from two different HIV-1 clades. In panel B, RNaseB, a folded-glycosylated protein (lane C) was treated as in panel A with purified proteasomes in the absence (lane P) or presence of epoxomicin (lane PI). The proteasomes were also unable to cleave RNaseB. The functional activity and the specificity of the proteasomes were demonstrated by the proteolytic cleavage of Gag-p24 (panel C, lane C) in the absence (lane P) or presence of epoxomicin (lane PI). Env-A244 and RNaseB (panels D and E, lanes C) were subjected to the same treatment as in panels A and B. Env-A244 and RNaseB were treated with Endo-F and Endo-H followed by incubation with purified proteasomes in the absence (lanes P) or presence of epoxomicin (lanes PI). Proteasomes were unable to cleave Env-A244 and RNaseB following treatment with either Endo-F or Endo-H. Env-A244 and RNaseB (panel F, lanes C) were reduced and alkylated (IAA) by treatment with iodioacetamine and DTT and then treated with purified proteasomes in the absence (lanes P) or presence of epoxomicin (lanes PI). Env-A244 remained resistant while RNaseB became susceptible to proteasomal cleavage. Samples were run on a 4–20% gradient Tris-glycine polyacrylamide gel. The molecular weight markers are shown in lane M in panels A, B, D, E, and F.
    Figure Legend Snippet: Proteasomes are unable to cleave HIV-1 Env proteins. Env-A244 (CRF01_AE) derived from CHO cells and gp140 (clade B) derived from H9 cells (panel A, lanes C) were treated with purified proteasomes isolated from activated CD4 + T-cells in the absence (lanes, P) or presence of epoxomicin, an irreversible proteasome inhibitor (lanes PI). Proteasomes were unable to cleave the Env proteins from two different HIV-1 clades. In panel B, RNaseB, a folded-glycosylated protein (lane C) was treated as in panel A with purified proteasomes in the absence (lane P) or presence of epoxomicin (lane PI). The proteasomes were also unable to cleave RNaseB. The functional activity and the specificity of the proteasomes were demonstrated by the proteolytic cleavage of Gag-p24 (panel C, lane C) in the absence (lane P) or presence of epoxomicin (lane PI). Env-A244 and RNaseB (panels D and E, lanes C) were subjected to the same treatment as in panels A and B. Env-A244 and RNaseB were treated with Endo-F and Endo-H followed by incubation with purified proteasomes in the absence (lanes P) or presence of epoxomicin (lanes PI). Proteasomes were unable to cleave Env-A244 and RNaseB following treatment with either Endo-F or Endo-H. Env-A244 and RNaseB (panel F, lanes C) were reduced and alkylated (IAA) by treatment with iodioacetamine and DTT and then treated with purified proteasomes in the absence (lanes P) or presence of epoxomicin (lanes PI). Env-A244 remained resistant while RNaseB became susceptible to proteasomal cleavage. Samples were run on a 4–20% gradient Tris-glycine polyacrylamide gel. The molecular weight markers are shown in lane M in panels A, B, D, E, and F.

    Techniques Used: Derivative Assay, Purification, Isolation, Functional Assay, Activity Assay, Incubation, Molecular Weight

    29) Product Images from "Trends in Thermostability Provide Information on the Nature of Substrate, Inhibitor, and Lipid Interactions with Mitochondrial Carriers *"

    Article Title: Trends in Thermostability Provide Information on the Nature of Substrate, Inhibitor, and Lipid Interactions with Mitochondrial Carriers *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.616607

    The relative thermal stability of mitochondrial carrier proteins. Carrier unfolding was monitored by the fluorescence of CPM-adduct formation at cysteine residues as they become solvent-exposed due to thermal denaturation (see “Materials and Methods”). A , thermal denaturation profile ( top ), and corresponding first derivative ( bottom ), of yeast AAC2 in assay buffer with 1.0% 12M in the absence ( solid line ) or presence ( dashed line ) of 20 μ m CATR. B , apparent T m of several AAC isoforms and human citrin in purification buffer (0.1% 12M, 50 m m NaCl, 10 m m Tris, pH 7.4, with 0.1 mg/ml tetraoleoyl cardiolipin) in the absence or presence of 20 μ m CATR, as indicated. Typical profiles are shown, and T m values are averages ± S.D. ( error bars ) of three tests. ScAAC2 (4 cysteines), MtAAC1 (2 cysteines), TlAAC (3 cysteines), CmAAC (2 cysteines), GsAAC (4 cysteines) and citrin (7 cysteines) are from S. cerevisiae , M. thermophila , T. lanuginosus , C. merolae , G. sulfuraria , and Homo sapiens , respectively.
    Figure Legend Snippet: The relative thermal stability of mitochondrial carrier proteins. Carrier unfolding was monitored by the fluorescence of CPM-adduct formation at cysteine residues as they become solvent-exposed due to thermal denaturation (see “Materials and Methods”). A , thermal denaturation profile ( top ), and corresponding first derivative ( bottom ), of yeast AAC2 in assay buffer with 1.0% 12M in the absence ( solid line ) or presence ( dashed line ) of 20 μ m CATR. B , apparent T m of several AAC isoforms and human citrin in purification buffer (0.1% 12M, 50 m m NaCl, 10 m m Tris, pH 7.4, with 0.1 mg/ml tetraoleoyl cardiolipin) in the absence or presence of 20 μ m CATR, as indicated. Typical profiles are shown, and T m values are averages ± S.D. ( error bars ) of three tests. ScAAC2 (4 cysteines), MtAAC1 (2 cysteines), TlAAC (3 cysteines), CmAAC (2 cysteines), GsAAC (4 cysteines) and citrin (7 cysteines) are from S. cerevisiae , M. thermophila , T. lanuginosus , C. merolae , G. sulfuraria , and Homo sapiens , respectively.

    Techniques Used: Fluorescence, Purification

    30) Product Images from "A New Platelet-Aggregation-Inhibiting Factor Isolated from Bothrops moojeni Snake Venom"

    Article Title: A New Platelet-Aggregation-Inhibiting Factor Isolated from Bothrops moojeni Snake Venom

    Journal: BioMed Research International

    doi: 10.1155/2017/4315832

    Purification of BmooPAi from B. moojeni snake venom. (a) Separation on DEAE-Sephacel ion-exchange chromatography: crude venom (200 mg) was applied to the column (2.5 × 20 cm) and elution was carried out at a flow rate of 20 mL/h with ammonium bicarbonate (Ambic) buffer gradients, pH 7.8, from 0.05 M to 0.6 M. Fractions of 3.0 mL/tube were collected and the absorbance was read at 280 nm. (b) Separation on Sephadex G-75 molecular exclusion chromatography: fraction D7 was applied to the column (1.0 × 100 cm) and elution with 0.05 M ammonium bicarbonate was achieved at a flow rate of 20 mL/h. Fractions of 3.0 mL/tube were collected and the absorbance was read at 280 nm. (c) Separation by affinity chromatography on a HiTrap Heparin HP column using the ÄKTApurifier HPLC system: fraction D7S2 was applied to the column (5 × 1 mL), previously equilibrated with 20 mM Tris-HCl buffer (pH 7.0) containing 5 mM calcium chloride. The samples were eluted with an increasing concentration gradient of 20 mM Tris-HCl buffer (pH 7.0) containing 2.0 M sodium chloride, and the absorbance of the fractions was monitored at 280 nm. Fractions of 1.0 mL/tube were collected at a flow rate of 30 mL/h. (d) SDS-PAGE in 14% (w/v) polyacrylamide, Tris-glycine buffer, pH 8.3, and 20 mA. Lanes: 1, standard proteins; 2, reduced crude venom of B. moojeni ; 3, reduced BmooPAi; 4, nonreduced BmooPAi. The gel was stained with Coomassie blue R-250. (e) Reverse-phase HPLC on a C2C18 column (4.6 × 100 mm) equilibrated with 0.1% trifluoroacetic acid (TFA) and eluted with a linear concentration gradient from 0 to 100% of solution B (70% acetonitrile in 0.1% TFA).
    Figure Legend Snippet: Purification of BmooPAi from B. moojeni snake venom. (a) Separation on DEAE-Sephacel ion-exchange chromatography: crude venom (200 mg) was applied to the column (2.5 × 20 cm) and elution was carried out at a flow rate of 20 mL/h with ammonium bicarbonate (Ambic) buffer gradients, pH 7.8, from 0.05 M to 0.6 M. Fractions of 3.0 mL/tube were collected and the absorbance was read at 280 nm. (b) Separation on Sephadex G-75 molecular exclusion chromatography: fraction D7 was applied to the column (1.0 × 100 cm) and elution with 0.05 M ammonium bicarbonate was achieved at a flow rate of 20 mL/h. Fractions of 3.0 mL/tube were collected and the absorbance was read at 280 nm. (c) Separation by affinity chromatography on a HiTrap Heparin HP column using the ÄKTApurifier HPLC system: fraction D7S2 was applied to the column (5 × 1 mL), previously equilibrated with 20 mM Tris-HCl buffer (pH 7.0) containing 5 mM calcium chloride. The samples were eluted with an increasing concentration gradient of 20 mM Tris-HCl buffer (pH 7.0) containing 2.0 M sodium chloride, and the absorbance of the fractions was monitored at 280 nm. Fractions of 1.0 mL/tube were collected at a flow rate of 30 mL/h. (d) SDS-PAGE in 14% (w/v) polyacrylamide, Tris-glycine buffer, pH 8.3, and 20 mA. Lanes: 1, standard proteins; 2, reduced crude venom of B. moojeni ; 3, reduced BmooPAi; 4, nonreduced BmooPAi. The gel was stained with Coomassie blue R-250. (e) Reverse-phase HPLC on a C2C18 column (4.6 × 100 mm) equilibrated with 0.1% trifluoroacetic acid (TFA) and eluted with a linear concentration gradient from 0 to 100% of solution B (70% acetonitrile in 0.1% TFA).

    Techniques Used: Purification, Ion Exchange Chromatography, Flow Cytometry, Chromatography, Affinity Chromatography, High Performance Liquid Chromatography, Concentration Assay, SDS Page, Staining

    31) Product Images from "Intrinsic Buffer Hydroxyl Radical Dosimetry Using Tris(Hydroxymethyl)Aminomethane"

    Article Title: Intrinsic Buffer Hydroxyl Radical Dosimetry Using Tris(Hydroxymethyl)Aminomethane

    Journal: bioRxiv

    doi: 10.1101/825463

    A) Tris absorbance change for myoglobin samples without MES scavenger, with 10 mM MES scavenger, and compensated conditions with 10 mM MES scavenger and increased laser fluence to obtain a ΔAbs 265 ≈ 4.97. B) (Blue) Peptide oxidation for myoglobin peptides in the absence of MES; (Orange) Peptide oxidation for myoglobin peptides in the presence of 10 mM MES; (Grey) Peptide oxidation for myoglobin in the presence of 10 mM MES under compensating laser fluence conditions, using Tris as a doseimeter for radical compensation. No statistically significant differences were detected in peptide oxidation between no MES samples and with MES-containing samples compensated using Tris dosimetry.
    Figure Legend Snippet: A) Tris absorbance change for myoglobin samples without MES scavenger, with 10 mM MES scavenger, and compensated conditions with 10 mM MES scavenger and increased laser fluence to obtain a ΔAbs 265 ≈ 4.97. B) (Blue) Peptide oxidation for myoglobin peptides in the absence of MES; (Orange) Peptide oxidation for myoglobin peptides in the presence of 10 mM MES; (Grey) Peptide oxidation for myoglobin in the presence of 10 mM MES under compensating laser fluence conditions, using Tris as a doseimeter for radical compensation. No statistically significant differences were detected in peptide oxidation between no MES samples and with MES-containing samples compensated using Tris dosimetry.

    Techniques Used:

    32) Product Images from "New Anti-Human Immunodeficiency Virus Type 1 6-Aminoquinolones: Mechanism of Action"

    Article Title: New Anti-Human Immunodeficiency Virus Type 1 6-Aminoquinolones: Mechanism of Action

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.47.3.889-896.2003

    Binding of WM5 to TAR RNA. Shown is the fraction of bound quinolone (ν) versus the TAR concentration for WM5 as inferred by fluorometric titrations (see text). Experiments were performed with a mixture of Tris-HCl (10 mM, pH 7.0), NaCl (20 mM), and Mg(ClO 4 ) 2 (1 mM) at 25°C.
    Figure Legend Snippet: Binding of WM5 to TAR RNA. Shown is the fraction of bound quinolone (ν) versus the TAR concentration for WM5 as inferred by fluorometric titrations (see text). Experiments were performed with a mixture of Tris-HCl (10 mM, pH 7.0), NaCl (20 mM), and Mg(ClO 4 ) 2 (1 mM) at 25°C.

    Techniques Used: Binding Assay, Concentration Assay

    33) Product Images from "Rapid Purification and Properties of Betaine Aldehyde Dehydrogenase from Pseudomonas aeruginosa"

    Article Title: Rapid Purification and Properties of Betaine Aldehyde Dehydrogenase from Pseudomonas aeruginosa

    Journal: Journal of Bacteriology

    doi:

    Effect of temperature, dilution, and buffer composition on the stability of BADH from P. aeruginosa . (A) Kinetics of the reversible heat inactivation. The enzyme (31 μg/ml) was incubated at 40°C in 10 mM potassium phosphate buffer (pH 8.0) containing 0.1 mM EDTA, 5 mM β-mercaptoethanol, 20% (wt/vol) sucrose, and 25 mM KCl in the absence (●) or presence (■) of 0.5 mM NADP + . After the times indicated, 4-μl samples were withdrawn and immediately added to the standard assay mixture. Reactivation of the heated enzyme (▴) was achieved by cooling the partially inactivated enzyme at 30°C. (B) Kinetics of the inactivation by dilution. Enzyme was diluted to 0.3 (●, ○, and ▴) or 3 (■ and □) μg/ml in 100 mM potassium phosphate buffer at pH 8.0 (●, ○, ■, and □) or pH 6.5 (▴) in the absence (●, ■, and ▴) or presence (○ and □) of 0.38 mM NADP + . The enzyme was incubated at 30°C and assayed at various time points to determine the activity remaining, using the standard assay. (C) Kinetics of inactivation in the absence of K + ions. Enzyme (31 μg/ml) was incubated at 30°C in 31 mM Tris-HCl buffer (pH 8.0) in the absence of KCl and NADP + (●) or in the presence of 25 mM KCl (○), 200 mM KCl (■), 0.5 mM NADP + (□), or 0.5 mM NADP + plus 25 mM KCl (▴) and was assayed at various time points to determine the activity remaining. In all panels the results are plotted as percentages of that for the untreated control. The lines are the results of the best fits of the experimental data to equation 4 (A) or to equation 5 (B and C).
    Figure Legend Snippet: Effect of temperature, dilution, and buffer composition on the stability of BADH from P. aeruginosa . (A) Kinetics of the reversible heat inactivation. The enzyme (31 μg/ml) was incubated at 40°C in 10 mM potassium phosphate buffer (pH 8.0) containing 0.1 mM EDTA, 5 mM β-mercaptoethanol, 20% (wt/vol) sucrose, and 25 mM KCl in the absence (●) or presence (■) of 0.5 mM NADP + . After the times indicated, 4-μl samples were withdrawn and immediately added to the standard assay mixture. Reactivation of the heated enzyme (▴) was achieved by cooling the partially inactivated enzyme at 30°C. (B) Kinetics of the inactivation by dilution. Enzyme was diluted to 0.3 (●, ○, and ▴) or 3 (■ and □) μg/ml in 100 mM potassium phosphate buffer at pH 8.0 (●, ○, ■, and □) or pH 6.5 (▴) in the absence (●, ■, and ▴) or presence (○ and □) of 0.38 mM NADP + . The enzyme was incubated at 30°C and assayed at various time points to determine the activity remaining, using the standard assay. (C) Kinetics of inactivation in the absence of K + ions. Enzyme (31 μg/ml) was incubated at 30°C in 31 mM Tris-HCl buffer (pH 8.0) in the absence of KCl and NADP + (●) or in the presence of 25 mM KCl (○), 200 mM KCl (■), 0.5 mM NADP + (□), or 0.5 mM NADP + plus 25 mM KCl (▴) and was assayed at various time points to determine the activity remaining. In all panels the results are plotted as percentages of that for the untreated control. The lines are the results of the best fits of the experimental data to equation 4 (A) or to equation 5 (B and C).

    Techniques Used: Incubation, Activity Assay

    34) Product Images from "Relationship between Prion Propensity and the Rates of Individual Molecular Steps of Fibril Assembly *"

    Article Title: Relationship between Prion Propensity and the Rates of Individual Molecular Steps of Fibril Assembly *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.208934

    Time courses of fibril formation in cross-seeding experiments monitored by ThT fluorescence. Solutions (20 μ m ) of Sc Ure2p ( A and B ) or Sp Ure2p ( C and D ) were incubated with a series of concentrations of Sc seeds ( A and C ) or Sp seeds ( B and D ): 0% seed ( brown ), 1% seed ( orange ), 2% seed ( ochre ), 4% seed ( green ), 7% seed ( teal ), 10% seed ( blue ), in 50 m m Tris-HCl, pH 8.4, 200 m m NaCl. The data were fitted globally to obtain the ratios of the rate constants for elongation and breakage, as described under “Experimental Procedures.”
    Figure Legend Snippet: Time courses of fibril formation in cross-seeding experiments monitored by ThT fluorescence. Solutions (20 μ m ) of Sc Ure2p ( A and B ) or Sp Ure2p ( C and D ) were incubated with a series of concentrations of Sc seeds ( A and C ) or Sp seeds ( B and D ): 0% seed ( brown ), 1% seed ( orange ), 2% seed ( ochre ), 4% seed ( green ), 7% seed ( teal ), 10% seed ( blue ), in 50 m m Tris-HCl, pH 8.4, 200 m m NaCl. The data were fitted globally to obtain the ratios of the rate constants for elongation and breakage, as described under “Experimental Procedures.”

    Techniques Used: Fluorescence, Incubation

    35) Product Images from "Targeting Anticancer Drug Delivery to Pancreatic Cancer Cells Using a Fucose-Bound Nanoparticle Approach"

    Article Title: Targeting Anticancer Drug Delivery to Pancreatic Cancer Cells Using a Fucose-Bound Nanoparticle Approach

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0039545

    Production and physicochemical properties of L-fucose-bound liposomes. ( A) Liposome preparation scheme showing sugar chains. HSA, BS 3 , Tris, and DTSSP denote the following, respectively: human serum albumin; bis(sulfosuccinimidyl) suberate; Tris(hydroxymethyl) aminomethane; 3,3-dithiobis (sulfosuccinimidylpropionate). ( B) Electron microscopic image of L-fucose-bound liposome. Scale bar shows 50 nm. ( C, D) Physicochemical characterization of Fuc-Liposome-Cy5.5. Average particle size (C) and zeta-potential (D) of liposomes that were prepared in water was determined by dynamic light scattering spectrophotometry.
    Figure Legend Snippet: Production and physicochemical properties of L-fucose-bound liposomes. ( A) Liposome preparation scheme showing sugar chains. HSA, BS 3 , Tris, and DTSSP denote the following, respectively: human serum albumin; bis(sulfosuccinimidyl) suberate; Tris(hydroxymethyl) aminomethane; 3,3-dithiobis (sulfosuccinimidylpropionate). ( B) Electron microscopic image of L-fucose-bound liposome. Scale bar shows 50 nm. ( C, D) Physicochemical characterization of Fuc-Liposome-Cy5.5. Average particle size (C) and zeta-potential (D) of liposomes that were prepared in water was determined by dynamic light scattering spectrophotometry.

    Techniques Used: Spectrophotometry

    36) Product Images from "Cation-induced shape programming and morphing in protein-based hydrogels"

    Article Title: Cation-induced shape programming and morphing in protein-based hydrogels

    Journal: Science Advances

    doi: 10.1126/sciadv.aba6112

    Programming of protein hydrogels with cations and morphing via chemomechanical changes. ( A ) BSA hydrogels were casted in cylindrical shape in PTFE tubes (top left) and programmed in a spring shape using a negative cast, by immersion in 2 M Zn 2+ solution (top right) or 1.5 M Cu 2+ solution (middle right) for 30 min; BSA hydrogels were produced in a flower shape using a silicone mold (bottom left) and programmed into a ring, by immersion in 2 M Zn 2+ solution for 30 min (bottom right). ( B ) Morphing from the programmed shape into the casted shape of BSA hydrogel upon immersion in regular Tris buffer at time 0 (left) and 5 min (right) for the hydrogels from (A). Movies S1 and S2 accompany this figure. (Photo credit: Luai R. Khoury, UWM; Marina Slawinski, UWM).
    Figure Legend Snippet: Programming of protein hydrogels with cations and morphing via chemomechanical changes. ( A ) BSA hydrogels were casted in cylindrical shape in PTFE tubes (top left) and programmed in a spring shape using a negative cast, by immersion in 2 M Zn 2+ solution (top right) or 1.5 M Cu 2+ solution (middle right) for 30 min; BSA hydrogels were produced in a flower shape using a silicone mold (bottom left) and programmed into a ring, by immersion in 2 M Zn 2+ solution for 30 min (bottom right). ( B ) Morphing from the programmed shape into the casted shape of BSA hydrogel upon immersion in regular Tris buffer at time 0 (left) and 5 min (right) for the hydrogels from (A). Movies S1 and S2 accompany this figure. (Photo credit: Luai R. Khoury, UWM; Marina Slawinski, UWM).

    Techniques Used: Produced

    37) Product Images from "Glyco-engineered Long Acting FGF21 Variant with Optimal Pharmaceutical and Pharmacokinetic Properties to Enable Weekly to Twice Monthly Subcutaneous Dosing"

    Article Title: Glyco-engineered Long Acting FGF21 Variant with Optimal Pharmaceutical and Pharmacokinetic Properties to Enable Weekly to Twice Monthly Subcutaneous Dosing

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-22456-w

    Formulation characterization of PF-06645849. ( a ) % HMMS formation at 25 °C in Tris pH 7.5 formulation for WT FGF21 (40 mg/mL), Fc-FGF21[N171] (100 mg/mL) and PF-06645849/Fc-FGF21 [R19V][N171] (100 mg/mL) over time. ( b ) % HMMS formation for PF-06645849 at pH 7.5/25 °C over time at concentrations of 61, 82, and 100 mg/mL, respectively. ( c ) Viscosity of PF-06645849/Fc-FGF21[R19V][N171] over concentration ranges. All experiments were carried out as n = 1.
    Figure Legend Snippet: Formulation characterization of PF-06645849. ( a ) % HMMS formation at 25 °C in Tris pH 7.5 formulation for WT FGF21 (40 mg/mL), Fc-FGF21[N171] (100 mg/mL) and PF-06645849/Fc-FGF21 [R19V][N171] (100 mg/mL) over time. ( b ) % HMMS formation for PF-06645849 at pH 7.5/25 °C over time at concentrations of 61, 82, and 100 mg/mL, respectively. ( c ) Viscosity of PF-06645849/Fc-FGF21[R19V][N171] over concentration ranges. All experiments were carried out as n = 1.

    Techniques Used: Concentration Assay

    38) Product Images from "Fibril growth and seeding capacity play key roles in α-synuclein-mediated apoptotic cell death"

    Article Title: Fibril growth and seeding capacity play key roles in α-synuclein-mediated apoptotic cell death

    Journal: Cell Death and Differentiation

    doi: 10.1038/cdd.2015.79

    Inhibiting fibril growth attenuates α -syn-induced toxicity in hippocampal primary neurons. Hippocampal primary culture were plated in 96-well plates and treated for 6 days with Tris buffer (negative control) or ( a ) the indicated α -syn
    Figure Legend Snippet: Inhibiting fibril growth attenuates α -syn-induced toxicity in hippocampal primary neurons. Hippocampal primary culture were plated in 96-well plates and treated for 6 days with Tris buffer (negative control) or ( a ) the indicated α -syn

    Techniques Used: Negative Control

    Mixture of α -syn monomers and fibrils but not individual α -syn species is toxic to M17 neuroblastoma cell line. ( a – g ) The human neuroblastoma M17 cells were treated with Tris buffer (50 mM Tris, pH 7.5, 150 mM NaCl;
    Figure Legend Snippet: Mixture of α -syn monomers and fibrils but not individual α -syn species is toxic to M17 neuroblastoma cell line. ( a – g ) The human neuroblastoma M17 cells were treated with Tris buffer (50 mM Tris, pH 7.5, 150 mM NaCl;

    Techniques Used:

    Mixture of α -syn monomers and fibrils initiates apoptosis in a caspase 8-dependent manner in hippocampal primary neurons. ( a – e ) Hippocampal primary neurons were plated in 96-well plate and treated for 6 days with Tris buffer (negative
    Figure Legend Snippet: Mixture of α -syn monomers and fibrils initiates apoptosis in a caspase 8-dependent manner in hippocampal primary neurons. ( a – e ) Hippocampal primary neurons were plated in 96-well plate and treated for 6 days with Tris buffer (negative

    Techniques Used:

    Mixture of α -syn monomers and fibrils exacerbates cell death in hippocampal primary neurons. ( a – f ) Hippocampal primary neurons were plated in 96-well plate and treated with Tris buffer or α -syn species for the indicated times.
    Figure Legend Snippet: Mixture of α -syn monomers and fibrils exacerbates cell death in hippocampal primary neurons. ( a – f ) Hippocampal primary neurons were plated in 96-well plate and treated with Tris buffer or α -syn species for the indicated times.

    Techniques Used:

    α -Syn mixture forms growing aggregates at the cell plasma membrane of hippocampal primary neurons. Hippocampal primary neurons were treated with Tris buffer (negative control) or α -syn species for the indicated times. ( a ) Orthogonal projections
    Figure Legend Snippet: α -Syn mixture forms growing aggregates at the cell plasma membrane of hippocampal primary neurons. Hippocampal primary neurons were treated with Tris buffer (negative control) or α -syn species for the indicated times. ( a ) Orthogonal projections

    Techniques Used: Negative Control

    39) Product Images from "Dynamics of the ?2-adrenergic G-protein coupled receptor revealed by hydrogen-deuterium exchange"

    Article Title: Dynamics of the ?2-adrenergic G-protein coupled receptor revealed by hydrogen-deuterium exchange

    Journal: Analytical chemistry

    doi: 10.1021/ac902484p

    Western blot analysis of carazolol-bound β 2 AR_460 after PNGase F incubation at 4°C for 1 hour, probed by using anti-FLAG (A), following separation by SDS-PAGE gel 10% Bis-Tris stained by SimplyBlue Safestain (B).
    Figure Legend Snippet: Western blot analysis of carazolol-bound β 2 AR_460 after PNGase F incubation at 4°C for 1 hour, probed by using anti-FLAG (A), following separation by SDS-PAGE gel 10% Bis-Tris stained by SimplyBlue Safestain (B).

    Techniques Used: Western Blot, Incubation, SDS Page, Staining

    40) Product Images from "Engineering the “Missing Link” in Biosynthetic (−)-Menthol Production: Bacterial Isopulegone Isomerase"

    Article Title: Engineering the “Missing Link” in Biosynthetic (−)-Menthol Production: Bacterial Isopulegone Isomerase

    Journal: ACS Catalysis

    doi: 10.1021/acscatal.7b04115

    Location of key active site mutations implicated in improving KSI activity toward 3 . (A) Residues located in the equilenin-binding region of wild type KSI from P. putida (PDB: 1OH0). 34 The residues and equilenin are shown as atom colored sticks with yellow and green carbons, respectively. Interactions are shown as red dotted lines. The backbone is shown as a gray cartoon. (B) Comparative steady-state activity of wild-type and variant KSI enzymes. Reaction mixtures (100 μL) were composed of 50 mM Tris pH 7.0 containing 1 mM 3 . The absorbance was monitored at 260 nm for 1 h at 20 °C. Inset: location of the variant residues in KSI V881/L99V/D103S. The backbone and mutations are shown as gray ribbons and balls, respectively (blue balls for V101).
    Figure Legend Snippet: Location of key active site mutations implicated in improving KSI activity toward 3 . (A) Residues located in the equilenin-binding region of wild type KSI from P. putida (PDB: 1OH0). 34 The residues and equilenin are shown as atom colored sticks with yellow and green carbons, respectively. Interactions are shown as red dotted lines. The backbone is shown as a gray cartoon. (B) Comparative steady-state activity of wild-type and variant KSI enzymes. Reaction mixtures (100 μL) were composed of 50 mM Tris pH 7.0 containing 1 mM 3 . The absorbance was monitored at 260 nm for 1 h at 20 °C. Inset: location of the variant residues in KSI V881/L99V/D103S. The backbone and mutations are shown as gray ribbons and balls, respectively (blue balls for V101).

    Techniques Used: Activity Assay, Binding Assay, Variant Assay

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    Real-time Polymerase Chain Reaction:

    Article Title: S100P enhances the motility and invasion of human trophoblast cell lines
    Article Snippet: .. qPCR analysis Following the appropriate incubation, trophoblast cells were collected after trypsinisation and mRNA extraction was carried out using TRIS reagent (Sigma, UK) according to the manufacturer’s protocol. qPCR was performed using S100P (sequences F: 5′-TCAAGGTGCTGATGGAGAA-3′ and R: 5′-ACACGATGAACTCACTGAA-3′) and β-actin primers (F: 5′-ATGTACGTTGCTATCCAGGC-3′ and R: 5′-CTCCTTAATGTCACGCACGAT-3′) and SYBR green mix (Roche, UK) according to the manufacturer’s protocols. ..

    Flow Cytometry:

    Article Title: Bacterially Expressed F1-20/AP-3 Assembles Clathrin Into Cages With a Narrow Size Distribution: Implications for the Regulation of Quantal Size During Neurotransmission
    Article Snippet: .. The Sepharose was then eluted three times with 0.5 ml 0.5 M Tris, 0.1 mM PMSF (pH 7.0) at 37°C for 15 min. Flow-through, washes, and eluates were all concentrated using Millipore quick concentrator-10s, and each sample was brought to 20 µl in IX SDS sample buffer. ..

    Buffer Exchange:

    Article Title: Cj1386, an Atypical Hemin-Binding Protein, Mediates Hemin Trafficking to KatA in Campylobacter jejuni
    Article Snippet: .. Buffer exchange with 100 mM NaCl, 20 mM Tris, pH 7.4, was performed using a 10-kDa-cutoff centrifugal filter unit (Millipore). ..

    SYBR Green Assay:

    Article Title: S100P enhances the motility and invasion of human trophoblast cell lines
    Article Snippet: .. qPCR analysis Following the appropriate incubation, trophoblast cells were collected after trypsinisation and mRNA extraction was carried out using TRIS reagent (Sigma, UK) according to the manufacturer’s protocol. qPCR was performed using S100P (sequences F: 5′-TCAAGGTGCTGATGGAGAA-3′ and R: 5′-ACACGATGAACTCACTGAA-3′) and β-actin primers (F: 5′-ATGTACGTTGCTATCCAGGC-3′ and R: 5′-CTCCTTAATGTCACGCACGAT-3′) and SYBR green mix (Roche, UK) according to the manufacturer’s protocols. ..

    Incubation:

    Article Title: S100P enhances the motility and invasion of human trophoblast cell lines
    Article Snippet: .. qPCR analysis Following the appropriate incubation, trophoblast cells were collected after trypsinisation and mRNA extraction was carried out using TRIS reagent (Sigma, UK) according to the manufacturer’s protocol. qPCR was performed using S100P (sequences F: 5′-TCAAGGTGCTGATGGAGAA-3′ and R: 5′-ACACGATGAACTCACTGAA-3′) and β-actin primers (F: 5′-ATGTACGTTGCTATCCAGGC-3′ and R: 5′-CTCCTTAATGTCACGCACGAT-3′) and SYBR green mix (Roche, UK) according to the manufacturer’s protocols. ..

    other:

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    Activity Assay:

    Article Title: Influence of sodium substitutes on 5-HT-mediated effects at mouse 5-HT3 receptors
    Article Snippet: .. The following compounds were used in the experiments (for chemical structures, see ): 5-hydroxytryptamine creatinine sulphate from Sigma, Munich, Germany; [14 C]guanidinium chloride (specific activity=59 mCi mmol−1 ) from CEA (Biotrend, Cologne, Germany); [3 H]GR65630, (specific activity 64.8 Ci mmol−1 ) from NEN DuPont (Dreieich, Germany); choline chloride, TMA chloride, Tris and NMDG were all from Sigma (Munich, Germany). .. Basal [ 14 C]guanidinium influx : In N1E-115 cells incubated in buffer containing 135 m M sodium, basal influx of [14 C]guanidinium, that is, influx in the absence of 5-HT, amounted to 16.0±0.5 pmol × mg protein−1 ( n =19).

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    Millipore htt shortstop 44q bis tris gel
    Mammalian-derived expanded <t>Htt</t> exon-1 and shortstop proteins form fibrillar structures as detected by transmission electron microscopy. Time course of GST-Htt exon-1 <t>44Q,</t> A , and GST-Htt shortstop 44Q, B , thrombin cleavage reaction as monitored by TEM.
    Htt Shortstop 44q Bis Tris Gel, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore tris
    Steady-state kinetics of ATP hydrolysis by the optimized catalytic domain. (A) The ATP hydrolysis is inhibited above 4 mM ATP concentration. (B) The hydrolysis of ATP by the enzyme shows a positive cooperativity up to 4 mM ATP concentration. The kinetics of hydrolysis was measured by following phosphate release in 10 mM <t>Tris,</t> pH = 7.6, 150 mM <t>NaCl,</t> and 1 mM Mg +2 at 37°C as described under Materials and Methods . Total protein concentration was 9.6 µg. Error bars correspond to standard deviation of triplicate measurements.
    Tris, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2261 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/tris/product/Millipore
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    Image Search Results


    Mammalian-derived expanded Htt exon-1 and shortstop proteins form fibrillar structures as detected by transmission electron microscopy. Time course of GST-Htt exon-1 44Q, A , and GST-Htt shortstop 44Q, B , thrombin cleavage reaction as monitored by TEM.

    Journal: The Journal of Biological Chemistry

    Article Title: Identification of Novel Potentially Toxic Oligomers Formed in Vitro from Mammalian-derived Expanded huntingtin Exon-1 Protein *

    doi: 10.1074/jbc.M111.252577

    Figure Lengend Snippet: Mammalian-derived expanded Htt exon-1 and shortstop proteins form fibrillar structures as detected by transmission electron microscopy. Time course of GST-Htt exon-1 44Q, A , and GST-Htt shortstop 44Q, B , thrombin cleavage reaction as monitored by TEM.

    Article Snippet: For Blue native-PAGE/Western blotting analysis, samples prepared in Blue native sample buffer were resolved on a 3–12% (Htt exon-1 44Q) or 4–16% (Htt shortstop 44Q) Bis-Tris gel and transferred to PVDF (Immobilon P, Millipore) using the Invitrogen system.

    Techniques: Derivative Assay, Transmission Assay, Electron Microscopy, Transmission Electron Microscopy

    Expanded Htt exon-1 and shortstop proteins form a variety of oligomeric and fibrillar aggregates as assessed by atomic force microscopy. A , quantification of numbers of oligomers and fibrils per unit area for both Htt exon-1 44Q and Htt shortstop 44Q

    Journal: The Journal of Biological Chemistry

    Article Title: Identification of Novel Potentially Toxic Oligomers Formed in Vitro from Mammalian-derived Expanded huntingtin Exon-1 Protein *

    doi: 10.1074/jbc.M111.252577

    Figure Lengend Snippet: Expanded Htt exon-1 and shortstop proteins form a variety of oligomeric and fibrillar aggregates as assessed by atomic force microscopy. A , quantification of numbers of oligomers and fibrils per unit area for both Htt exon-1 44Q and Htt shortstop 44Q

    Article Snippet: For Blue native-PAGE/Western blotting analysis, samples prepared in Blue native sample buffer were resolved on a 3–12% (Htt exon-1 44Q) or 4–16% (Htt shortstop 44Q) Bis-Tris gel and transferred to PVDF (Immobilon P, Millipore) using the Invitrogen system.

    Techniques: Microscopy

    Expanded Htt exon-1 protein generated in mammalian cells forms SDS-stable and native oligomeric complexes. Time course of GST-Htt exon-1 44Q thrombin cleavage reaction resolved by SDS-PAGE, A , or Blue native-PAGE, B , followed by Western blotting. A , an

    Journal: The Journal of Biological Chemistry

    Article Title: Identification of Novel Potentially Toxic Oligomers Formed in Vitro from Mammalian-derived Expanded huntingtin Exon-1 Protein *

    doi: 10.1074/jbc.M111.252577

    Figure Lengend Snippet: Expanded Htt exon-1 protein generated in mammalian cells forms SDS-stable and native oligomeric complexes. Time course of GST-Htt exon-1 44Q thrombin cleavage reaction resolved by SDS-PAGE, A , or Blue native-PAGE, B , followed by Western blotting. A , an

    Article Snippet: For Blue native-PAGE/Western blotting analysis, samples prepared in Blue native sample buffer were resolved on a 3–12% (Htt exon-1 44Q) or 4–16% (Htt shortstop 44Q) Bis-Tris gel and transferred to PVDF (Immobilon P, Millipore) using the Invitrogen system.

    Techniques: Generated, SDS Page, Blue Native PAGE, Western Blot

    Htt-derived oligomeric complexes are not detected by SDS- or Blue native-PAGE for expanded Htt shortstop protein. Time course of GST-Htt shortstop 44Q thrombin cleavage reaction resolved by SDS-PAGE, A , or Blue native-PAGE, B , followed by Western blotting.

    Journal: The Journal of Biological Chemistry

    Article Title: Identification of Novel Potentially Toxic Oligomers Formed in Vitro from Mammalian-derived Expanded huntingtin Exon-1 Protein *

    doi: 10.1074/jbc.M111.252577

    Figure Lengend Snippet: Htt-derived oligomeric complexes are not detected by SDS- or Blue native-PAGE for expanded Htt shortstop protein. Time course of GST-Htt shortstop 44Q thrombin cleavage reaction resolved by SDS-PAGE, A , or Blue native-PAGE, B , followed by Western blotting.

    Article Snippet: For Blue native-PAGE/Western blotting analysis, samples prepared in Blue native sample buffer were resolved on a 3–12% (Htt exon-1 44Q) or 4–16% (Htt shortstop 44Q) Bis-Tris gel and transferred to PVDF (Immobilon P, Millipore) using the Invitrogen system.

    Techniques: Derivative Assay, Blue Native PAGE, SDS Page, Western Blot

    Steady-state kinetics of ATP hydrolysis by the optimized catalytic domain. (A) The ATP hydrolysis is inhibited above 4 mM ATP concentration. (B) The hydrolysis of ATP by the enzyme shows a positive cooperativity up to 4 mM ATP concentration. The kinetics of hydrolysis was measured by following phosphate release in 10 mM Tris, pH = 7.6, 150 mM NaCl, and 1 mM Mg +2 at 37°C as described under Materials and Methods . Total protein concentration was 9.6 µg. Error bars correspond to standard deviation of triplicate measurements.

    Journal: PLoS ONE

    Article Title: Identification of Small-Molecule Inhibitors of Yersinia pestis Type III Secretion System YscN ATPase

    doi: 10.1371/journal.pone.0019716

    Figure Lengend Snippet: Steady-state kinetics of ATP hydrolysis by the optimized catalytic domain. (A) The ATP hydrolysis is inhibited above 4 mM ATP concentration. (B) The hydrolysis of ATP by the enzyme shows a positive cooperativity up to 4 mM ATP concentration. The kinetics of hydrolysis was measured by following phosphate release in 10 mM Tris, pH = 7.6, 150 mM NaCl, and 1 mM Mg +2 at 37°C as described under Materials and Methods . Total protein concentration was 9.6 µg. Error bars correspond to standard deviation of triplicate measurements.

    Article Snippet: The protein fractions were buffer exchanged into 10 mM Tris, pH 7.6, 150 mM NaCl, 10% glycerol and concentrated to 0.6–0.9 mg/ml using the Amicon Ultra-15 concentrators (Millipore).

    Techniques: Concentration Assay, Protein Concentration, Standard Deviation

    The bacterially expressed 33 kD NH 2 -terminus of F1-20/AP-3 binds specifically to clathrin triskelia; 15 µg of the bacterially expressed 33 kD NH 2 -terminus of F1-20/AP-3 was incubated with 0.5 ml clathrin-Sepharose in 0.5 ml isolation buffer at 4°C for 2 hr ( A ), and binding was monitored by batch analysis, as described in Methods. Fraction 1 is the flow-through; fractions 2,3,4 are washes with isolation buffer; and fractions 5,6,7 are eluates with 0.5 M Tris (pH 7.0). All samples were analyzed by SDS-PAGE, followed by silver staining. Negative controls were carried out by incubating 15 µg bacterially expressed 33 kD NH 2 -terminus of F1-20/AP-3 with 0.5 ml underivatized Sepharose ( B ), and by incubating 15 µg E. coli GST protein with 0.5 ml clathrin-Sepharose ( C ).

    Journal: Journal of neuroscience research

    Article Title: Bacterially Expressed F1-20/AP-3 Assembles Clathrin Into Cages With a Narrow Size Distribution: Implications for the Regulation of Quantal Size During Neurotransmission

    doi: 10.1002/jnr.490410104

    Figure Lengend Snippet: The bacterially expressed 33 kD NH 2 -terminus of F1-20/AP-3 binds specifically to clathrin triskelia; 15 µg of the bacterially expressed 33 kD NH 2 -terminus of F1-20/AP-3 was incubated with 0.5 ml clathrin-Sepharose in 0.5 ml isolation buffer at 4°C for 2 hr ( A ), and binding was monitored by batch analysis, as described in Methods. Fraction 1 is the flow-through; fractions 2,3,4 are washes with isolation buffer; and fractions 5,6,7 are eluates with 0.5 M Tris (pH 7.0). All samples were analyzed by SDS-PAGE, followed by silver staining. Negative controls were carried out by incubating 15 µg bacterially expressed 33 kD NH 2 -terminus of F1-20/AP-3 with 0.5 ml underivatized Sepharose ( B ), and by incubating 15 µg E. coli GST protein with 0.5 ml clathrin-Sepharose ( C ).

    Article Snippet: The Sepharose was then eluted three times with 0.5 ml 0.5 M Tris, 0.1 mM PMSF (pH 7.0) at 37°C for 15 min. Flow-through, washes, and eluates were all concentrated using Millipore quick concentrator-10s, and each sample was brought to 20 µl in IX SDS sample buffer.

    Techniques: Incubation, Isolation, Binding Assay, Flow Cytometry, SDS Page, Silver Staining

    Tyrosine 57 is important for hemin affinity to Cj1386. (A) Absorption spectra of 10 μM Cj1386 WT and 10 μM Cj1386 Y57A in 100 mM NaCl, 20 mM Tris, pH 7.4. (B) Absorption spectra of 10 μM Cj1386 Y57A and 10 μM Cj1386 Y57A plus

    Journal: Journal of Bacteriology

    Article Title: Cj1386, an Atypical Hemin-Binding Protein, Mediates Hemin Trafficking to KatA in Campylobacter jejuni

    doi: 10.1128/JB.02346-14

    Figure Lengend Snippet: Tyrosine 57 is important for hemin affinity to Cj1386. (A) Absorption spectra of 10 μM Cj1386 WT and 10 μM Cj1386 Y57A in 100 mM NaCl, 20 mM Tris, pH 7.4. (B) Absorption spectra of 10 μM Cj1386 Y57A and 10 μM Cj1386 Y57A plus

    Article Snippet: Buffer exchange with 100 mM NaCl, 20 mM Tris, pH 7.4, was performed using a 10-kDa-cutoff centrifugal filter unit (Millipore).

    Techniques:

    Y57A Cj1386 can be reconstituted with hemin and displays 1:1 hemin-binding stoichiometry. (A) Absorption spectra of Cj1386 Y57A when titrated with hemin at 1 μM increments against 10 μM apo-Cj1386 Y57A in 100 mM NaCl, 20 mM Tris, pH 7.4.

    Journal: Journal of Bacteriology

    Article Title: Cj1386, an Atypical Hemin-Binding Protein, Mediates Hemin Trafficking to KatA in Campylobacter jejuni

    doi: 10.1128/JB.02346-14

    Figure Lengend Snippet: Y57A Cj1386 can be reconstituted with hemin and displays 1:1 hemin-binding stoichiometry. (A) Absorption spectra of Cj1386 Y57A when titrated with hemin at 1 μM increments against 10 μM apo-Cj1386 Y57A in 100 mM NaCl, 20 mM Tris, pH 7.4.

    Article Snippet: Buffer exchange with 100 mM NaCl, 20 mM Tris, pH 7.4, was performed using a 10-kDa-cutoff centrifugal filter unit (Millipore).

    Techniques: Binding Assay