tris  (Millipore)

 
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  • 97
    Name:
    Tris hydroxymethyl aminomethane
    Description:

    Catalog Number:
    154563
    Price:
    None
    Applications:
    Buffer and primary pH standard. pKa=8.3 at 20C. Auxiliary material in pharmaceutical science.
    Buy from Supplier


    Structured Review

    Millipore tris
    Tris hydroxymethyl aminomethane

    https://www.bioz.com/result/tris/product/Millipore
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    tris - by Bioz Stars, 2021-07
    97/100 stars

    Images

    1) 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:

    2) 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

    3) Product Images from "Principal Component Analysis Reveals Age-Related and Muscle-Type-Related Differences in Protein Carbonyl Profiles of Muscle Mitochondria"

    Article Title: Principal Component Analysis Reveals Age-Related and Muscle-Type-Related Differences in Protein Carbonyl Profiles of Muscle Mitochondria

    Journal: The journals of gerontology. Series A, Biological sciences and medical sciences

    doi:

    Electropherograms of mitochondrial proteins labeled with Alexa 488 hydrazide. A, Young, fast-twitch. B, Old, fast-twitch. C, Young, slow-twitch. D, Old, slow-twitch. Separation, −570 V/cm; hydrodynamic injection, 11 kPa, 4 seconds; sieving matrix, 20 mM Tris, 20 mM tricine, 8% dextran (462 kD), 0.5% sodium dodecyl sulfate, pH 8. The samples were analyzed in triplicate. Capillary conditioning, fluorescence labeling, and detection are described in Materials and Methods. A.U. = arbitrary units.
    Figure Legend Snippet: Electropherograms of mitochondrial proteins labeled with Alexa 488 hydrazide. A, Young, fast-twitch. B, Old, fast-twitch. C, Young, slow-twitch. D, Old, slow-twitch. Separation, −570 V/cm; hydrodynamic injection, 11 kPa, 4 seconds; sieving matrix, 20 mM Tris, 20 mM tricine, 8% dextran (462 kD), 0.5% sodium dodecyl sulfate, pH 8. The samples were analyzed in triplicate. Capillary conditioning, fluorescence labeling, and detection are described in Materials and Methods. A.U. = arbitrary units.

    Techniques Used: Labeling, Injection, Fluorescence

    4) Product Images from "Compositional adaptability in NPM1-SURF6 scaffolding networks enabled by dynamic switching of phase separation mechanisms"

    Article Title: Compositional adaptability in NPM1-SURF6 scaffolding networks enabled by dynamic switching of phase separation mechanisms

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07530-1

    Homotypic NPM1 droplets undergo reversible aging. a NPM1 concentrations measured within the dense (green, n ≥ 343 droplets) and light (black, n = 6) phases prepared in the presence of 5%, 15% and 30% PEG (see Methods). Values represent mean ± s.d. b Confocal microscopy images of NPM1-A488 droplets photobleached at t = 75 min (top row), 120 min (middle row), and 210 min (bottom row) after mixing 20 μM NPM1 with 5% PEG in 10 mM Tris, 150 mM NaCl, 2 mM DTT, pH 7.5 buffer; scale bar = 1 μm. c FRAP recovery curves of NPM1-A488 in 20 μM NPM1 droplets at t = 75 min (blue), 120 min (magenta), and 210 min (orange) after droplet formation in the presence of 5% PEG (left panel) and 15% PEG (right panel). Values represent mean ± s.d. for n ≥ 8 droplets. Under both crowding agent conditions, the fraction of NPM1 that recovered decreased at the later time points after mixing; ROI = 1 µm circular area in the center of the droplet. d Time-lapse fluorescence microscopy images of fusion between NPM1-A488 droplets formed 60 min (top row) or 180 min (bottom row) after mixing in the presence of 5% PEG. Droplets fused rapidly after incubation for 60 min, but fusion was very slow after incubation for 180 min. e An illustration of the experimental scheme used to monitor dissolution of aged NPM1 droplets formed in 5% PEG upon removal of the crowding agent. Light phase (90% of the solution) was gently removed and replaced with the same volume of buffer lacking PEG. Droplet dissolution was monitored over time using confocal fluorescence microscopy imaging. f Time-lapse imaging of aged droplets dissolving after removal of the crowding agent
    Figure Legend Snippet: Homotypic NPM1 droplets undergo reversible aging. a NPM1 concentrations measured within the dense (green, n ≥ 343 droplets) and light (black, n = 6) phases prepared in the presence of 5%, 15% and 30% PEG (see Methods). Values represent mean ± s.d. b Confocal microscopy images of NPM1-A488 droplets photobleached at t = 75 min (top row), 120 min (middle row), and 210 min (bottom row) after mixing 20 μM NPM1 with 5% PEG in 10 mM Tris, 150 mM NaCl, 2 mM DTT, pH 7.5 buffer; scale bar = 1 μm. c FRAP recovery curves of NPM1-A488 in 20 μM NPM1 droplets at t = 75 min (blue), 120 min (magenta), and 210 min (orange) after droplet formation in the presence of 5% PEG (left panel) and 15% PEG (right panel). Values represent mean ± s.d. for n ≥ 8 droplets. Under both crowding agent conditions, the fraction of NPM1 that recovered decreased at the later time points after mixing; ROI = 1 µm circular area in the center of the droplet. d Time-lapse fluorescence microscopy images of fusion between NPM1-A488 droplets formed 60 min (top row) or 180 min (bottom row) after mixing in the presence of 5% PEG. Droplets fused rapidly after incubation for 60 min, but fusion was very slow after incubation for 180 min. e An illustration of the experimental scheme used to monitor dissolution of aged NPM1 droplets formed in 5% PEG upon removal of the crowding agent. Light phase (90% of the solution) was gently removed and replaced with the same volume of buffer lacking PEG. Droplet dissolution was monitored over time using confocal fluorescence microscopy imaging. f Time-lapse imaging of aged droplets dissolving after removal of the crowding agent

    Techniques Used: Confocal Microscopy, Fluorescence, Microscopy, Incubation, Imaging

    5) 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

    6) Product Images from "Stabilization of Dry Sucrose Glasses by Four LEA_4 Proteins from Arabidopsis thaliana"

    Article Title: Stabilization of Dry Sucrose Glasses by Four LEA_4 Proteins from Arabidopsis thaliana

    Journal: Biomolecules

    doi: 10.3390/biom11050615

    Wavenumber-temperature coefficients in the glassy state (WTC g ) of dry Suc. Samples contained either pure Suc or, in addition, the proteins LEA11, COR15A, LEA25, LEA29, β-lactoglobulin (LG) or bovine serum albumin (BSA). All solutions contained 10 mg/mL Suc and were prepared in H 2 O ( A ), 10 mM Tris ( B ) or 10 mM NaP buffer (pH 7.4) ( C ). In addition, samples contained proteins at the indicated Suc/protein mass ratios. Samples were dried on CaF 2 windows. The bars indicate the means from at least three samples ± standard deviation. Statistically significant differences between the values determined at the two different Suc/LEA mass ratios are indicated by *, while significant differences between the values obtained in the presence of the different proteins and pure Suc are indicated by + (*, + ˂0.05; **, ++ ˂0.01, ***, +++ ˂0.001).
    Figure Legend Snippet: Wavenumber-temperature coefficients in the glassy state (WTC g ) of dry Suc. Samples contained either pure Suc or, in addition, the proteins LEA11, COR15A, LEA25, LEA29, β-lactoglobulin (LG) or bovine serum albumin (BSA). All solutions contained 10 mg/mL Suc and were prepared in H 2 O ( A ), 10 mM Tris ( B ) or 10 mM NaP buffer (pH 7.4) ( C ). In addition, samples contained proteins at the indicated Suc/protein mass ratios. Samples were dried on CaF 2 windows. The bars indicate the means from at least three samples ± standard deviation. Statistically significant differences between the values determined at the two different Suc/LEA mass ratios are indicated by *, while significant differences between the values obtained in the presence of the different proteins and pure Suc are indicated by + (*, + ˂0.05; **, ++ ˂0.01, ***, +++ ˂0.001).

    Techniques Used: Standard Deviation

    Normalized amide I peaks from FTIR spectra of dry LEA11 ( A – C ), COR15A ( D – F ), LEA25 ( G – I ) and LEA29 ( J – L ). Samples contained the LEA proteins in the absence or presence of Suc at a mass ratio 2 in H 2 O (no additive) ( A , D , G , J ), 10 mM Tris ( B , E , H , K ), or 10 mM NaP (pH 7.4) ( C , F , I , L ) as indicated in the panels. Samples were dried on CaF 2 windows, and spectra were recorded at 30 °C.
    Figure Legend Snippet: Normalized amide I peaks from FTIR spectra of dry LEA11 ( A – C ), COR15A ( D – F ), LEA25 ( G – I ) and LEA29 ( J – L ). Samples contained the LEA proteins in the absence or presence of Suc at a mass ratio 2 in H 2 O (no additive) ( A , D , G , J ), 10 mM Tris ( B , E , H , K ), or 10 mM NaP (pH 7.4) ( C , F , I , L ) as indicated in the panels. Samples were dried on CaF 2 windows, and spectra were recorded at 30 °C.

    Techniques Used:

    Melting curves of dry Suc glasses determined from the temperature-induced shift of the position of the νOH peak in FTIR spectra. Suc at a final concentration of 10 mg/mL was dissolved in pure H 2 O, in Tris or NaP buffer (10 mM, pH 7.4) and dried on CaF 2 windows. Glass transition temperatures (T g ) were determined from the intersection of fitted regression lines in the glassy state at low and the melted state at higher temperatures. T g of the different samples is indicated.
    Figure Legend Snippet: Melting curves of dry Suc glasses determined from the temperature-induced shift of the position of the νOH peak in FTIR spectra. Suc at a final concentration of 10 mg/mL was dissolved in pure H 2 O, in Tris or NaP buffer (10 mM, pH 7.4) and dried on CaF 2 windows. Glass transition temperatures (T g ) were determined from the intersection of fitted regression lines in the glassy state at low and the melted state at higher temperatures. T g of the different samples is indicated.

    Techniques Used: Concentration Assay

    FTIR spectra of dry Suc glass in the fingerprint region (1500–1150 cm −1 ) at 30 °C. Samples contained 10 mg/mL Suc in pure H 2 O, or in 10 mM Tris or 10 mM NaP (pH 7.4). Samples were dried on CaF 2 windows, and spectra were recorded at 30 °C. Spectra were normalized to the absorbance at 1420 cm −1 . The vibration peak at about 1268 cm −1 is indicated with an arrow.
    Figure Legend Snippet: FTIR spectra of dry Suc glass in the fingerprint region (1500–1150 cm −1 ) at 30 °C. Samples contained 10 mg/mL Suc in pure H 2 O, or in 10 mM Tris or 10 mM NaP (pH 7.4). Samples were dried on CaF 2 windows, and spectra were recorded at 30 °C. Spectra were normalized to the absorbance at 1420 cm −1 . The vibration peak at about 1268 cm −1 is indicated with an arrow.

    Techniques Used:

    Glass transition temperature (T g ) of dry Suc. Samples contained either pure Suc or, in addition, the proteins LEA11, COR15A, LEA25, LEA29, β-lactoglobulin (LG) or bovine serum albumin (BSA). All solutions contained 10 mg/mL Suc and were prepared in H 2 O ( A ), 10 mM Tris ( B ) or 10 mM NaP buffer (pH 7.4) ( C ). In addition, samples contained proteins at the indicated Suc/protein mass ratios. Samples were dried on CaF 2 windows. The bars indicate the means from at least three samples ± standard deviation. Statistically significant differences between the values determined at the two different Suc/LEA mass ratios are indicated by *, while significant differences between the values obtained in the presence of the different proteins and pure Suc are indicated by + (*, + ˂0.05; **, ++ ˂0.01, +++ ˂0.001).
    Figure Legend Snippet: Glass transition temperature (T g ) of dry Suc. Samples contained either pure Suc or, in addition, the proteins LEA11, COR15A, LEA25, LEA29, β-lactoglobulin (LG) or bovine serum albumin (BSA). All solutions contained 10 mg/mL Suc and were prepared in H 2 O ( A ), 10 mM Tris ( B ) or 10 mM NaP buffer (pH 7.4) ( C ). In addition, samples contained proteins at the indicated Suc/protein mass ratios. Samples were dried on CaF 2 windows. The bars indicate the means from at least three samples ± standard deviation. Statistically significant differences between the values determined at the two different Suc/LEA mass ratios are indicated by *, while significant differences between the values obtained in the presence of the different proteins and pure Suc are indicated by + (*, + ˂0.05; **, ++ ˂0.01, +++ ˂0.001).

    Techniques Used: Standard Deviation

    Influence of the presence of buffers and proteins on the position of the absorbance peak located at about 1268 cm −1 in a pure Suc glass. Samples contained either pure Suc or, in addition, the proteins LEA11, COR15A, LEA25, LEA29, β-lactoglobulin (LG) or bovine serum albumin (BSA). All solutions contained 10 mg/mL Suc and were prepared in H 2 O ( A ), 10 mM Tris ( B ) or 10 mM NaP buffer (pH 7.4) ( C ). Samples were dried on CaF 2 windows, and spectra were recorded at 30 °C. Values are means from at least three parallel samples ± standard deviation. Statistically significant differences between the values determined at the two different Suc/LEA mass ratios are indicated by *, while significant differences between the values obtained in the presence of the different proteins and pure Suc are indicated by + (*, + ˂0.05; **, ++ ˂0.01, ***, +++ ˂0.001).
    Figure Legend Snippet: Influence of the presence of buffers and proteins on the position of the absorbance peak located at about 1268 cm −1 in a pure Suc glass. Samples contained either pure Suc or, in addition, the proteins LEA11, COR15A, LEA25, LEA29, β-lactoglobulin (LG) or bovine serum albumin (BSA). All solutions contained 10 mg/mL Suc and were prepared in H 2 O ( A ), 10 mM Tris ( B ) or 10 mM NaP buffer (pH 7.4) ( C ). Samples were dried on CaF 2 windows, and spectra were recorded at 30 °C. Values are means from at least three parallel samples ± standard deviation. Statistically significant differences between the values determined at the two different Suc/LEA mass ratios are indicated by *, while significant differences between the values obtained in the presence of the different proteins and pure Suc are indicated by + (*, + ˂0.05; **, ++ ˂0.01, ***, +++ ˂0.001).

    Techniques Used: Standard Deviation

    Position of the νOH peak of dry Suc in the glassy state at 30 °C. Samples contained either pure Suc or, in addition, the proteins LEA11, COR15A, LEA25, LEA29, β-lactoglobulin (LG) or bovine serum albumin (BSA). All solutions contained 10 mg/mL Suc and were prepared in H 2 O ( A ), 10 mM Tris ( B ) or 10 mM NaP buffer (pH 7.4) ( C ). In addition, samples contained proteins at the indicated Suc/protein mass ratios. Samples were dried on CaF 2 windows. The bars indicate the means from at least three samples ± standard deviation. Statistically significant differences between the values determined at the two different Suc/LEA mass ratios are indicated by *, while significant differences between the values obtained in the presence of the different proteins and pure Suc are indicated by + ( +
    Figure Legend Snippet: Position of the νOH peak of dry Suc in the glassy state at 30 °C. Samples contained either pure Suc or, in addition, the proteins LEA11, COR15A, LEA25, LEA29, β-lactoglobulin (LG) or bovine serum albumin (BSA). All solutions contained 10 mg/mL Suc and were prepared in H 2 O ( A ), 10 mM Tris ( B ) or 10 mM NaP buffer (pH 7.4) ( C ). In addition, samples contained proteins at the indicated Suc/protein mass ratios. Samples were dried on CaF 2 windows. The bars indicate the means from at least three samples ± standard deviation. Statistically significant differences between the values determined at the two different Suc/LEA mass ratios are indicated by *, while significant differences between the values obtained in the presence of the different proteins and pure Suc are indicated by + ( +

    Techniques Used: Standard Deviation

    7) Product Images from "Molecular architecture of E. coli purine nucleoside phosphorylase studied by analytical ultracentrifugation and CD spectroscopy"

    Article Title: Molecular architecture of E. coli purine nucleoside phosphorylase studied by analytical ultracentrifugation and CD spectroscopy

    Journal: Protein Science : A Publication of the Protein Society

    doi: 10.1110/ps.062183206

    Sedimentation coefficient distributions ( g ( s 0 20,w )) obtained for E. coli PNP . Sedimentation velocity experiments were carried out in ( A ) 50 mM Tris-HCl (pH 7.0) for 1.3 mg/mL (– –) and 0.03 mg/mL (–) PNP and in 50 mM phosphate buffer (pH 7.0) with 1M GdnHCl for 0.6 mg/mL (- - -) and 0.03 mg/mL (○) PNP and ( B ) for 1.3 mg/mL PNP in 50 mM Tris-HCl (pH 7.0) without (– –) and with (–) 0.5 M NaCl, with 50 mM sodium phosphate (○), or with 0.5 M NaCl and 50 mM sodium phosphate (- - -). Centrifugation was at 60,000 rpm and 20°C.
    Figure Legend Snippet: Sedimentation coefficient distributions ( g ( s 0 20,w )) obtained for E. coli PNP . Sedimentation velocity experiments were carried out in ( A ) 50 mM Tris-HCl (pH 7.0) for 1.3 mg/mL (– –) and 0.03 mg/mL (–) PNP and in 50 mM phosphate buffer (pH 7.0) with 1M GdnHCl for 0.6 mg/mL (- - -) and 0.03 mg/mL (○) PNP and ( B ) for 1.3 mg/mL PNP in 50 mM Tris-HCl (pH 7.0) without (– –) and with (–) 0.5 M NaCl, with 50 mM sodium phosphate (○), or with 0.5 M NaCl and 50 mM sodium phosphate (- - -). Centrifugation was at 60,000 rpm and 20°C.

    Techniques Used: Sedimentation, Centrifugation

    Sedimentation equilibrium experiment of E. coli PNP in 50 mM Tris-HCl buffer (pH 7.5) with 100 mM KCl and 0.1 mM DTT at 20°C. Centrifugation was performed at 10,200 rpm. The enzyme was loaded at 0.46 mg/mL ( A ), 1.08 mg/mL ( B ), and 1.7 mg/mL ( C ). Protein concentration gradient in equilibrium measured at 280 nm (shown as open circles at bottom ) is shown together with the single-species model fit (line). ( Top ) Residuals (A exp –A mod ) for this model (fitted simultaneously to all three data sets). Similar curves and residual plots were obtained for the self-association model (i.e., coexistence of monomers and N-mer oligomer). The following parameters for these two models were obtained: single-species model, M = 150,746 Da; self-association model, M monomer = 25,988 Da, N = 5.8, K a = 4.0 × 10 87 M −5 .
    Figure Legend Snippet: Sedimentation equilibrium experiment of E. coli PNP in 50 mM Tris-HCl buffer (pH 7.5) with 100 mM KCl and 0.1 mM DTT at 20°C. Centrifugation was performed at 10,200 rpm. The enzyme was loaded at 0.46 mg/mL ( A ), 1.08 mg/mL ( B ), and 1.7 mg/mL ( C ). Protein concentration gradient in equilibrium measured at 280 nm (shown as open circles at bottom ) is shown together with the single-species model fit (line). ( Top ) Residuals (A exp –A mod ) for this model (fitted simultaneously to all three data sets). Similar curves and residual plots were obtained for the self-association model (i.e., coexistence of monomers and N-mer oligomer). The following parameters for these two models were obtained: single-species model, M = 150,746 Da; self-association model, M monomer = 25,988 Da, N = 5.8, K a = 4.0 × 10 87 M −5 .

    Techniques Used: Sedimentation, Centrifugation, Protein Concentration

    8) 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

    9) Product Images from "Heme ligand identification and redox properties of the cytochrome c synthetase, CcmF"

    Article Title: Heme ligand identification and redox properties of the cytochrome c synthetase, CcmF

    Journal: Biochemistry

    doi: 10.1021/bi201508t

    Redox titration of the CcmF b -heme. Spectra collected during a typical reductive titration of CcmF b -heme with nile blue chloride (A) and the corresponding linear Nernst plot (B). Arrows in (A) indicate the direction of changes in absorption during the course of the titration. In (B), [25 mV ln ( b -heme red / b -heme ox )] was used for the one-electron reduction of heme and [12.5 mV ln (dye red /dye ox )] was used for the two-electron reduction of dye, where b heme red / b -heme ox and dye red /dye ox represent ratios of the molar concentrations of the reduced and oxidized forms of the b -heme and the dye, respectively. Conditions: 20 mM Tris-HCl, pH 7, 100 mM NaCl, 0.02 % DDM.
    Figure Legend Snippet: Redox titration of the CcmF b -heme. Spectra collected during a typical reductive titration of CcmF b -heme with nile blue chloride (A) and the corresponding linear Nernst plot (B). Arrows in (A) indicate the direction of changes in absorption during the course of the titration. In (B), [25 mV ln ( b -heme red / b -heme ox )] was used for the one-electron reduction of heme and [12.5 mV ln (dye red /dye ox )] was used for the two-electron reduction of dye, where b heme red / b -heme ox and dye red /dye ox represent ratios of the molar concentrations of the reduced and oxidized forms of the b -heme and the dye, respectively. Conditions: 20 mM Tris-HCl, pH 7, 100 mM NaCl, 0.02 % DDM.

    Techniques Used: Titration

    Soret-excited resonance Raman spectra of the heme b in ferric (red) and ferrous (violet, blue) CcmF. Sample solutions were 88 μM in holoCcmF, 20 mM in Tris, pH 8, 100mM in NaCl, 0.02 % in dodecyl maltoside, ~ 2 nm in imidazole. HoloCcmF concentrations in the 3.2 % and 0.48 % DDM samples were 23 and 25 μM, respectively. The red and violet spectra were recorded using 10 mW of laser light at 413.1 nm (line focus of emission from Kr + laser). The low-frequency blue spectrum was recorded with 2 mW of 441.6-nm emission from a HeCd laser to identify the Fe−His stretching band.
    Figure Legend Snippet: Soret-excited resonance Raman spectra of the heme b in ferric (red) and ferrous (violet, blue) CcmF. Sample solutions were 88 μM in holoCcmF, 20 mM in Tris, pH 8, 100mM in NaCl, 0.02 % in dodecyl maltoside, ~ 2 nm in imidazole. HoloCcmF concentrations in the 3.2 % and 0.48 % DDM samples were 23 and 25 μM, respectively. The red and violet spectra were recorded using 10 mW of laser light at 413.1 nm (line focus of emission from Kr + laser). The low-frequency blue spectrum was recorded with 2 mW of 441.6-nm emission from a HeCd laser to identify the Fe−His stretching band.

    Techniques Used:

    10) 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

    11) 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

    12) 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

    13) 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

    14) Product Images from "Bacterial Expression of Human Butyrylcholinesterase as a Tool for Nerve Agent Bioscavengers Development"

    Article Title: Bacterial Expression of Human Butyrylcholinesterase as a Tool for Nerve Agent Bioscavengers Development

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

    doi: 10.3390/molecules22111828

    Oligomeric state analysis of pure hBChE-7. After separation of pure hBChE-7 on Superdex 200 Increase 10/300 equilibrated in 20 mM Tris pH 8.0, 150 mM NaCl, the major UV peak (plain line) was analyzed in line by multi-angles light scattering and a constant molecular weight of 70 + 2 kDa was measured (dashed line).
    Figure Legend Snippet: Oligomeric state analysis of pure hBChE-7. After separation of pure hBChE-7 on Superdex 200 Increase 10/300 equilibrated in 20 mM Tris pH 8.0, 150 mM NaCl, the major UV peak (plain line) was analyzed in line by multi-angles light scattering and a constant molecular weight of 70 + 2 kDa was measured (dashed line).

    Techniques Used: Molecular Weight

    15) 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

    16) Product Images from "Fatty Acids are Key in 4-Hydroxy-2-Nonenal-Mediated Activation of Uncoupling Proteins 1 and 2"

    Article Title: Fatty Acids are Key in 4-Hydroxy-2-Nonenal-Mediated Activation of Uncoupling Proteins 1 and 2

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0077786

    Analysis of HNE-mediated UCP1 modification. A. Membrane conductance decrease in the presence of Cys, Lys, and His blockers. Membranes were made from E. polar lipid (1mg/ml) and reconstituted with arachidonic acid (15 mol%) and UCP1 (charge 18, 11.9 µg/(mg of lipid). Bar 1 shows the conductance of the membrane without additives (control). NEM, NHS and MNBS in concentrations of 0.5 µM, 1.3 µM and 0.2 µM respectively were incubated with protein as described in Results. HNE was added in concentration 860 µM (grey bars). Buffer solution contained 50 mM Na 2 SO 4 , 10 mM Tris, 10 mM MES, 0.6 mM EGTA at pH 7.5. Data points represent mean ± standard deviation from 3–5 independent experiments. * p
    Figure Legend Snippet: Analysis of HNE-mediated UCP1 modification. A. Membrane conductance decrease in the presence of Cys, Lys, and His blockers. Membranes were made from E. polar lipid (1mg/ml) and reconstituted with arachidonic acid (15 mol%) and UCP1 (charge 18, 11.9 µg/(mg of lipid). Bar 1 shows the conductance of the membrane without additives (control). NEM, NHS and MNBS in concentrations of 0.5 µM, 1.3 µM and 0.2 µM respectively were incubated with protein as described in Results. HNE was added in concentration 860 µM (grey bars). Buffer solution contained 50 mM Na 2 SO 4 , 10 mM Tris, 10 mM MES, 0.6 mM EGTA at pH 7.5. Data points represent mean ± standard deviation from 3–5 independent experiments. * p

    Techniques Used: Modification, Incubation, Concentration Assay, Standard Deviation

    The effect of 4-hydroxy-2-nonenal on membrane conductance (G). A. Influence of HNE and/or arachidonic acid (AA) on G in the absence (white bars) and presence of UCP1 (grey bars) or UCP2 (dark grey bars). The concentrations of E. coli polar lipid, UCP1, UCP2 and HNE were 1 mg/ml, 5.5 µg/(mg of lipid, charge 9), 4.7 µg/(mg of lipid) and 0.86 mM respectively. AA was directly added at a concentration of 15 mol% to the lipid phase prior the membrane formation. The buffer solution contained 50 mM K 2 SO 4 , 25 mM TES 0.6 mM EGTA at pH 7.4 and T = 32°C. HNE was directly added to the buffer solution. Data points represent mean ± standard deviation from 3–5 independent experiments. B. Comparison of membrane conductance ratios in the presence (G) and absence (G 0 ) of 0.64 mM HNE for arachidic (ArA, 20:0), linoleic (LA, 18:2, ω6), eicosatrienoic (EA, 20:3, ω3) and arachidonic acid (AA, 20:4, ω6). Inset. Membrane conductance in the presence (grey bars) and absence (black bars) of HNE. Membranes from E. polar lipid were reconstituted with 15 mol% FA and 7 µg UCP1/(mg of lipid) (charge 33). The buffer solution contained 50 mM Na 2 SO 4 , 10 mM Tris, 10 mM MES, 0.6 mM EGTA, at pH 7.4 and T = 32°C. Data represent mean ± standard deviation from at least 3 independent experiments.
    Figure Legend Snippet: The effect of 4-hydroxy-2-nonenal on membrane conductance (G). A. Influence of HNE and/or arachidonic acid (AA) on G in the absence (white bars) and presence of UCP1 (grey bars) or UCP2 (dark grey bars). The concentrations of E. coli polar lipid, UCP1, UCP2 and HNE were 1 mg/ml, 5.5 µg/(mg of lipid, charge 9), 4.7 µg/(mg of lipid) and 0.86 mM respectively. AA was directly added at a concentration of 15 mol% to the lipid phase prior the membrane formation. The buffer solution contained 50 mM K 2 SO 4 , 25 mM TES 0.6 mM EGTA at pH 7.4 and T = 32°C. HNE was directly added to the buffer solution. Data points represent mean ± standard deviation from 3–5 independent experiments. B. Comparison of membrane conductance ratios in the presence (G) and absence (G 0 ) of 0.64 mM HNE for arachidic (ArA, 20:0), linoleic (LA, 18:2, ω6), eicosatrienoic (EA, 20:3, ω3) and arachidonic acid (AA, 20:4, ω6). Inset. Membrane conductance in the presence (grey bars) and absence (black bars) of HNE. Membranes from E. polar lipid were reconstituted with 15 mol% FA and 7 µg UCP1/(mg of lipid) (charge 33). The buffer solution contained 50 mM Na 2 SO 4 , 10 mM Tris, 10 mM MES, 0.6 mM EGTA, at pH 7.4 and T = 32°C. Data represent mean ± standard deviation from at least 3 independent experiments.

    Techniques Used: Concentration Assay, Standard Deviation, Acetylene Reduction Assay

    Dependence of UCP-mediated proton conductance on HNE concentration. Membranes from E. polar lipid were reconstituted with 15% arachidonic acid and 10.5 µg/(mg lipid) UCP1 (charge 19). Buffer solution contained 50 mM Na 2 SO 4 , 10 mM Tris, 10 mM MES, 0,6 mM EGTA, at pH 7.5 and T = 32°C. Data points represent mean ± standard deviation from at least 3 independent experiments. The data points were fitted following Michaelis-Menten kinetics as described in the text.
    Figure Legend Snippet: Dependence of UCP-mediated proton conductance on HNE concentration. Membranes from E. polar lipid were reconstituted with 15% arachidonic acid and 10.5 µg/(mg lipid) UCP1 (charge 19). Buffer solution contained 50 mM Na 2 SO 4 , 10 mM Tris, 10 mM MES, 0,6 mM EGTA, at pH 7.5 and T = 32°C. Data points represent mean ± standard deviation from at least 3 independent experiments. The data points were fitted following Michaelis-Menten kinetics as described in the text.

    Techniques Used: Concentration Assay, Standard Deviation

    17) 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

    18) 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

    19) 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

    20) 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

    21) Product Images from "Compositional adaptability in NPM1-SURF6 scaffolding networks enabled by dynamic switching of phase separation mechanisms"

    Article Title: Compositional adaptability in NPM1-SURF6 scaffolding networks enabled by dynamic switching of phase separation mechanisms

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07530-1

    Homotypic NPM1 droplets undergo reversible aging. a NPM1 concentrations measured within the dense (green, n ≥ 343 droplets) and light (black, n = 6) phases prepared in the presence of 5%, 15% and 30% PEG (see Methods). Values represent mean ± s.d. b Confocal microscopy images of NPM1-A488 droplets photobleached at t = 75 min (top row), 120 min (middle row), and 210 min (bottom row) after mixing 20 μM NPM1 with 5% PEG in 10 mM Tris, 150 mM NaCl, 2 mM DTT, pH 7.5 buffer; scale bar = 1 μm. c FRAP recovery curves of NPM1-A488 in 20 μM NPM1 droplets at t = 75 min (blue), 120 min (magenta), and 210 min (orange) after droplet formation in the presence of 5% PEG (left panel) and 15% PEG (right panel). Values represent mean ± s.d. for n ≥ 8 droplets. Under both crowding agent conditions, the fraction of NPM1 that recovered decreased at the later time points after mixing; ROI = 1 µm circular area in the center of the droplet. d Time-lapse fluorescence microscopy images of fusion between NPM1-A488 droplets formed 60 min (top row) or 180 min (bottom row) after mixing in the presence of 5% PEG. Droplets fused rapidly after incubation for 60 min, but fusion was very slow after incubation for 180 min. e An illustration of the experimental scheme used to monitor dissolution of aged NPM1 droplets formed in 5% PEG upon removal of the crowding agent. Light phase (90% of the solution) was gently removed and replaced with the same volume of buffer lacking PEG. Droplet dissolution was monitored over time using confocal fluorescence microscopy imaging. f Time-lapse imaging of aged droplets dissolving after removal of the crowding agent
    Figure Legend Snippet: Homotypic NPM1 droplets undergo reversible aging. a NPM1 concentrations measured within the dense (green, n ≥ 343 droplets) and light (black, n = 6) phases prepared in the presence of 5%, 15% and 30% PEG (see Methods). Values represent mean ± s.d. b Confocal microscopy images of NPM1-A488 droplets photobleached at t = 75 min (top row), 120 min (middle row), and 210 min (bottom row) after mixing 20 μM NPM1 with 5% PEG in 10 mM Tris, 150 mM NaCl, 2 mM DTT, pH 7.5 buffer; scale bar = 1 μm. c FRAP recovery curves of NPM1-A488 in 20 μM NPM1 droplets at t = 75 min (blue), 120 min (magenta), and 210 min (orange) after droplet formation in the presence of 5% PEG (left panel) and 15% PEG (right panel). Values represent mean ± s.d. for n ≥ 8 droplets. Under both crowding agent conditions, the fraction of NPM1 that recovered decreased at the later time points after mixing; ROI = 1 µm circular area in the center of the droplet. d Time-lapse fluorescence microscopy images of fusion between NPM1-A488 droplets formed 60 min (top row) or 180 min (bottom row) after mixing in the presence of 5% PEG. Droplets fused rapidly after incubation for 60 min, but fusion was very slow after incubation for 180 min. e An illustration of the experimental scheme used to monitor dissolution of aged NPM1 droplets formed in 5% PEG upon removal of the crowding agent. Light phase (90% of the solution) was gently removed and replaced with the same volume of buffer lacking PEG. Droplet dissolution was monitored over time using confocal fluorescence microscopy imaging. f Time-lapse imaging of aged droplets dissolving after removal of the crowding agent

    Techniques Used: Confocal Microscopy, Fluorescence, Microscopy, Incubation, Imaging

    22) Product Images from "Purification and characterization of glutathione reductase (E.C. 1.8.1.7) from bovine filarial worms Setaria cervi"

    Article Title: Purification and characterization of glutathione reductase (E.C. 1.8.1.7) from bovine filarial worms Setaria cervi

    Journal: Journal of Parasitic Diseases: Official Organ of the Indian Society for Parasitology

    doi: 10.1007/s12639-012-0138-8

    Elution profile for total protein and total activity of S. cervi GR from DEAE-Sepharose column. W1–W2 1.0 ml fractions after washing the column with Tris buffer, 1–13 1.0 ml fractions (proteins) eluted by applying a linear gradient of NaCl from 0 to 1.0 M in starting buffer. Activity was expressed as μmol NADP released/min ± SD based on experiments done in quadruplicates
    Figure Legend Snippet: Elution profile for total protein and total activity of S. cervi GR from DEAE-Sepharose column. W1–W2 1.0 ml fractions after washing the column with Tris buffer, 1–13 1.0 ml fractions (proteins) eluted by applying a linear gradient of NaCl from 0 to 1.0 M in starting buffer. Activity was expressed as μmol NADP released/min ± SD based on experiments done in quadruplicates

    Techniques Used: Activity Assay

    23) 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

    24) Product Images from "Control of Recombination Directionality by the Listeria Phage A118 Protein Gp44 and the Coiled-Coil Motif of Its Serine Integrase"

    Article Title: Control of Recombination Directionality by the Listeria Phage A118 Protein Gp44 and the Coiled-Coil Motif of Its Serine Integrase

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00019-17

    Properties of Gp44. (A) MALDI-TOF MS analysis of purified native Gp44. The calculated mass of the 71-residue polypeptide lacking an N-terminal methionine is 8,442 Da. (B) Circular dichroism of Gp44. Data were measured at 25°C in 50 mM PO 4 (pH 7.0) at a protein concentration of 22 μM and increasing concentrations of n -propanol to promote secondary structure. (C) Sedimentation equilibrium (SE) analysis was performed at three speeds and two concentrations at 4°C. Linearized data for a single speed and concentration are shown here, where scale = 2 RT /[ M (1 − v ̄ ρ)ω 2 ], where R is 1.98 kcal/mol, T is temperature, and M is molar mass. Other symbols are defined in Materials and Methods. M and D indicate the calculated data lines expected for single species monomer and dimer, respectively. The upwards curvature in the experimental data is indicative of an associating system. Global fit of the radial distributions gives an estimate of 13 μM for the dimer K d (see Materials and Methods). (D) Sedimentation velocity (SV) analysis of Gp44. A 44 μM monomer was sedimented at 42,000 rpm at 4°C. Shown is the distribution of species derived from the fitting of the data with the Lamm equation. (E) Size exclusion chromatography on a Superdex 75 10/300 column at 25°C, where Gp44 was injected at 246 μM. Dashed gray lines represent standards. The analyses presented in panels C to E were performed in 20 mM Tris (pH 7.5), 300 mM NaCl.
    Figure Legend Snippet: Properties of Gp44. (A) MALDI-TOF MS analysis of purified native Gp44. The calculated mass of the 71-residue polypeptide lacking an N-terminal methionine is 8,442 Da. (B) Circular dichroism of Gp44. Data were measured at 25°C in 50 mM PO 4 (pH 7.0) at a protein concentration of 22 μM and increasing concentrations of n -propanol to promote secondary structure. (C) Sedimentation equilibrium (SE) analysis was performed at three speeds and two concentrations at 4°C. Linearized data for a single speed and concentration are shown here, where scale = 2 RT /[ M (1 − v ̄ ρ)ω 2 ], where R is 1.98 kcal/mol, T is temperature, and M is molar mass. Other symbols are defined in Materials and Methods. M and D indicate the calculated data lines expected for single species monomer and dimer, respectively. The upwards curvature in the experimental data is indicative of an associating system. Global fit of the radial distributions gives an estimate of 13 μM for the dimer K d (see Materials and Methods). (D) Sedimentation velocity (SV) analysis of Gp44. A 44 μM monomer was sedimented at 42,000 rpm at 4°C. Shown is the distribution of species derived from the fitting of the data with the Lamm equation. (E) Size exclusion chromatography on a Superdex 75 10/300 column at 25°C, where Gp44 was injected at 246 μM. Dashed gray lines represent standards. The analyses presented in panels C to E were performed in 20 mM Tris (pH 7.5), 300 mM NaCl.

    Techniques Used: Mass Spectrometry, Purification, Protein Concentration, Sedimentation, Concentration Assay, Derivative Assay, Size-exclusion Chromatography, Injection

    25) Product Images from "Rationally designed oral vaccines can set an evolutionary trap for Salmonella Typhimurium"

    Article Title: Rationally designed oral vaccines can set an evolutionary trap for Salmonella Typhimurium

    Journal: bioRxiv

    doi: 10.1101/824821

    Synthetic and natural deletions of wzyB reduce the fitness of S . Tm in presence of Tris-EDTA and serum complement. The deletion of wzyB does not affect the growth of S .Tm or S .Tm Δ oafA Δ gtrC in LB (No stress) (A) but impairs growth in presence of Tris-EDTA (B) . Dashed lines represent the range of variations between experiments. This was in line with the outcome of competitions between S .Tm expressing constitutive Green Fluorescent Protein ( S .Tm GFP ) and S .Tm Δ oafA Δ gtrC Δ wzyB or a wzyB mutant isolated from an Evoltrap vaccinated mouse (C) . The level of GFP corrected for the optical density (OD) served as readout to quantify the S .Tm GFP population at the end of the overnight growth, in presence of S.Tm Δ oafA Δ gtrC , S.Tm Δ oafA Δ gtrC Δ wzyB or an evolved S .Tm Δ wzyB , in LB with or without Tris-EDTA. Values above 1 (dashed line) indicates that relatively more GFP was detected in presence of Tris-EDTA than without, which resulted from a competitive advantage of S .Tm GFP in presence of stress. D . The deletion of wzyB makes Salmonella sensitive to human serum complement. Values below 10 0 (dashed line) indicates that the number of colony-forming units (CFU) detected after incubation in human serum was lower than after incubation in heat inactivated human serum.
    Figure Legend Snippet: Synthetic and natural deletions of wzyB reduce the fitness of S . Tm in presence of Tris-EDTA and serum complement. The deletion of wzyB does not affect the growth of S .Tm or S .Tm Δ oafA Δ gtrC in LB (No stress) (A) but impairs growth in presence of Tris-EDTA (B) . Dashed lines represent the range of variations between experiments. This was in line with the outcome of competitions between S .Tm expressing constitutive Green Fluorescent Protein ( S .Tm GFP ) and S .Tm Δ oafA Δ gtrC Δ wzyB or a wzyB mutant isolated from an Evoltrap vaccinated mouse (C) . The level of GFP corrected for the optical density (OD) served as readout to quantify the S .Tm GFP population at the end of the overnight growth, in presence of S.Tm Δ oafA Δ gtrC , S.Tm Δ oafA Δ gtrC Δ wzyB or an evolved S .Tm Δ wzyB , in LB with or without Tris-EDTA. Values above 1 (dashed line) indicates that relatively more GFP was detected in presence of Tris-EDTA than without, which resulted from a competitive advantage of S .Tm GFP in presence of stress. D . The deletion of wzyB makes Salmonella sensitive to human serum complement. Values below 10 0 (dashed line) indicates that the number of colony-forming units (CFU) detected after incubation in human serum was lower than after incubation in heat inactivated human serum.

    Techniques Used: Expressing, Mutagenesis, Isolation, Incubation

    26) Product Images from "A continuous spectrophotometric assay for human cystathionine beta-synthase"

    Article Title: A continuous spectrophotometric assay for human cystathionine beta-synthase

    Journal: Analytical biochemistry

    doi: 10.1016/j.ab.2005.03.051

    (A) Response of the coupled UV assay to hCBS concentration. Shown are the primary data for NADH oxidation with time, under typical assay conditions [30 mM L-serine, 30 mM cysteamine, 1 mM MgCl 2 , 2 mM PEP, 0.1 mM PLP, PEPC (0.45 U), LDC (0.52 U), and MDH (2.1 U) in 200 mM Tris buffer, pH 8.0] with increasing hCBS concentration [ rate increases in the order: 0 (control; open triangles), 0.7 (filled circles), 1.4 (open squares), 2.1 (filled triangles), 2.8 (open diamonds), and 3.5 μg (filled diamonds); all in a 100-μL final volume]. (B) Data replotted as rate vs [CBS].
    Figure Legend Snippet: (A) Response of the coupled UV assay to hCBS concentration. Shown are the primary data for NADH oxidation with time, under typical assay conditions [30 mM L-serine, 30 mM cysteamine, 1 mM MgCl 2 , 2 mM PEP, 0.1 mM PLP, PEPC (0.45 U), LDC (0.52 U), and MDH (2.1 U) in 200 mM Tris buffer, pH 8.0] with increasing hCBS concentration [ rate increases in the order: 0 (control; open triangles), 0.7 (filled circles), 1.4 (open squares), 2.1 (filled triangles), 2.8 (open diamonds), and 3.5 μg (filled diamonds); all in a 100-μL final volume]. (B) Data replotted as rate vs [CBS].

    Techniques Used: UV Assay, Concentration Assay, Plasmid Purification

    27) Product Images from "SARS‐CoV‐2 nucleocapsid protein phase‐separates with RNA and with human hnRNPs"

    Article Title: SARS‐CoV‐2 nucleocapsid protein phase‐separates with RNA and with human hnRNPs

    Journal: The EMBO Journal

    doi: 10.15252/embj.2020106478

    The N‐terminal and linker intrinsically disordered domains and the C‐terminal dimerization domain are essential for robust N LLPS in vitro DIC micrographs of 50 μM N and domain deletion variants in 50 mM Tris 70 mM NaCl pH 7.4 without and with TEV protease (to cleave MBP from N) and without or with 0.5 mg/ml desalted total torula yeast RNA. Scale bars represent 80 μm. Phase separation over time as monitored by turbidity of 50 μM full‐length N or deletion variants after addition of TEV protease in the absence (B) or presence (C) of 0.5 mg/ml desalted total torula yeast RNA. Error bars represent standard deviation of three replicates.
    Figure Legend Snippet: The N‐terminal and linker intrinsically disordered domains and the C‐terminal dimerization domain are essential for robust N LLPS in vitro DIC micrographs of 50 μM N and domain deletion variants in 50 mM Tris 70 mM NaCl pH 7.4 without and with TEV protease (to cleave MBP from N) and without or with 0.5 mg/ml desalted total torula yeast RNA. Scale bars represent 80 μm. Phase separation over time as monitored by turbidity of 50 μM full‐length N or deletion variants after addition of TEV protease in the absence (B) or presence (C) of 0.5 mg/ml desalted total torula yeast RNA. Error bars represent standard deviation of three replicates.

    Techniques Used: In Vitro, Standard Deviation

    SARS‐CoV‐2 N phase separation enhanced by non‐specific RNA binding Turbidity of N is increased in the presence of torula yeast RNA and homopolymeric RNAs. Error bars represent standard deviation of three replicates. DIC micrographs of MBP‐N in the presence of TEV (to cleave MBP from N to initiate LLPS) with indicated RNA. After cleavage of MBP, N phase separates. Apparent polyG RNA induces aggregation of MBP‐N. Sample conditions: 50 µM MBP‐N, 0.5 mg/ml RNA/polyX, 70 mM NaCl, 25˚C, 50 mM Tris pH 7.4. Scale bar represents 80 µm.
    Figure Legend Snippet: SARS‐CoV‐2 N phase separation enhanced by non‐specific RNA binding Turbidity of N is increased in the presence of torula yeast RNA and homopolymeric RNAs. Error bars represent standard deviation of three replicates. DIC micrographs of MBP‐N in the presence of TEV (to cleave MBP from N to initiate LLPS) with indicated RNA. After cleavage of MBP, N phase separates. Apparent polyG RNA induces aggregation of MBP‐N. Sample conditions: 50 µM MBP‐N, 0.5 mg/ml RNA/polyX, 70 mM NaCl, 25˚C, 50 mM Tris pH 7.4. Scale bar represents 80 µm.

    Techniques Used: RNA Binding Assay, Standard Deviation

    SARS‐CoV‐2 N LLPS is modulated by salt and RNA 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 (A) with varying torula yeast RNA (at 100 mM NaCl) or (B) varying NaCl concentrations (at constant RNA concentration). Error bars represent standard deviation of three replicates. DIC micrographs of 50 μM MBP‐N in 50 mM Tris pH 7.4 (C) with varying torula yeast RNA concentrations (at 100 mM sodium chloride) and (D) varying sodium chloride concentrations (at constant RNA concentration), with or 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 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 (A) with varying torula yeast RNA (at 100 mM NaCl) or (B) varying NaCl concentrations (at constant RNA concentration). Error bars represent standard deviation of three replicates. DIC micrographs of 50 μM MBP‐N in 50 mM Tris pH 7.4 (C) with varying torula yeast RNA concentrations (at 100 mM sodium chloride) and (D) varying sodium chloride concentrations (at constant RNA concentration), with or without TEV protease (to cleave MBP from N). Scale bars represent 50 μm.

    Techniques Used: Concentration Assay, Standard Deviation

    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 the presence of salt (183 mM NaCl) and 0.3 mg/ml RNA at pH 7.4 (50 mM Tris), pH 6.1, pH 5.5, or pH 4.9 (pH 6.1 and below in 20 mM MES). Error bars represent standard deviation of three replicates. For samples at (B) pH 7.4 or (C) pH 5.5, DIC micrographs of 50 μM MBP‐N in the presence of salt (183 mM NaCl) without or with TEV protease (to cleave MBP from N) and without or with 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 the presence of salt (183 mM NaCl) and 0.3 mg/ml RNA at pH 7.4 (50 mM Tris), pH 6.1, pH 5.5, or pH 4.9 (pH 6.1 and below in 20 mM MES). Error bars represent standard deviation of three replicates. For samples at (B) pH 7.4 or (C) pH 5.5, DIC micrographs of 50 μM MBP‐N in the presence of salt (183 mM NaCl) without or with TEV protease (to cleave MBP from N) and without or with 0.3 mg/ml desalted total torula yeast RNA. Scale bars represent 50 μm.

    Techniques Used: Standard Deviation

    28) 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

    29) 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

    30) 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

    31) Product Images from "Effects of Length and Loop Composition on Structural Diversity and Similarity of (G3TG3NmG3TG3) G-Quadruplexes"

    Article Title: Effects of Length and Loop Composition on Structural Diversity and Similarity of (G3TG3NmG3TG3) G-Quadruplexes

    Journal: Molecules

    doi: 10.3390/molecules25081779

    The effect of a single base change in loop 1 and 3 on G4 formation of (G 3 HG 3 N m G 3 HG 3 )-FN sequences. ( a ) NMR spectra of A16T-FN, A10A-FN, and C10C-FN in 10 mM Tris and after 1 h and overnight (O/N) addition of 20 mM K + together with their PAGE assays of marker bands of HT24 (T 2 AG 3 ) 4 , HT48 (T 2 AG 3 ) 8 , and HT96 (T 2 AG 3 ) 16 and each sequence after 1h and O/N addition of 20 mM K + for A16T-FN, A10A-FN, and C10C-FN. ( b ) NMR spectra of T4T-FN, A4A-FN, and C4C-FN in 10 mM Tris and after 1 h and O/N addition of 20 mM K + together with their PAGE assays of marker bands of HT24 (T 2 AG 3 ) 4 , HT48 (T 2 AG 3 ) 8 , and HT96 (T 2 AG 3 ) 16 and each sequence after 1h and O/N addition of 20 mM K + for T4T-FN, A4A-FN, and C4C-FN. The same DNA concentration of 100 μM was used in the experiments of NMR and PAGE of this work.
    Figure Legend Snippet: The effect of a single base change in loop 1 and 3 on G4 formation of (G 3 HG 3 N m G 3 HG 3 )-FN sequences. ( a ) NMR spectra of A16T-FN, A10A-FN, and C10C-FN in 10 mM Tris and after 1 h and overnight (O/N) addition of 20 mM K + together with their PAGE assays of marker bands of HT24 (T 2 AG 3 ) 4 , HT48 (T 2 AG 3 ) 8 , and HT96 (T 2 AG 3 ) 16 and each sequence after 1h and O/N addition of 20 mM K + for A16T-FN, A10A-FN, and C10C-FN. ( b ) NMR spectra of T4T-FN, A4A-FN, and C4C-FN in 10 mM Tris and after 1 h and O/N addition of 20 mM K + together with their PAGE assays of marker bands of HT24 (T 2 AG 3 ) 4 , HT48 (T 2 AG 3 ) 8 , and HT96 (T 2 AG 3 ) 16 and each sequence after 1h and O/N addition of 20 mM K + for T4T-FN, A4A-FN, and C4C-FN. The same DNA concentration of 100 μM was used in the experiments of NMR and PAGE of this work.

    Techniques Used: Nuclear Magnetic Resonance, Polyacrylamide Gel Electrophoresis, Marker, Sequencing, Concentration Assay

    The effect of loop length on G4 formation of (G 3 TG 3 N m G 3 TG 3 ) sequences. CD and NMR spectra of T16T, T10T, T4T, T2T, and TTT in 10 mM Tris and after 1 h and overnight (O/N) addition of 100 mM K + together with their PAGE assays of marker bands of HT24 (T 2 AG 3 ) 4 , HT48 (T 2 AG 3 ) 8 , and HT96 (T 2 AG 3 ) 16 (lane 1) and each sequence after 1 h and O/N addition of 100 mM K + for T16T, T10T, T4T, T2T, and TTT. The same DNA concentration of 100 μM was used in the experiments of CD, NMR and PAGE of this work.
    Figure Legend Snippet: The effect of loop length on G4 formation of (G 3 TG 3 N m G 3 TG 3 ) sequences. CD and NMR spectra of T16T, T10T, T4T, T2T, and TTT in 10 mM Tris and after 1 h and overnight (O/N) addition of 100 mM K + together with their PAGE assays of marker bands of HT24 (T 2 AG 3 ) 4 , HT48 (T 2 AG 3 ) 8 , and HT96 (T 2 AG 3 ) 16 (lane 1) and each sequence after 1 h and O/N addition of 100 mM K + for T16T, T10T, T4T, T2T, and TTT. The same DNA concentration of 100 μM was used in the experiments of CD, NMR and PAGE of this work.

    Techniques Used: Nuclear Magnetic Resonance, Polyacrylamide Gel Electrophoresis, Marker, Sequencing, Concentration Assay

    The Effect on G4 formation of native G-rich sequences without flanking nucleotides. CD and NMR spectra of GTERT-d(FN), PIG4-d(FN), and chl1 -d(FN) in 10 mM Tris and after 1 h and overnight (O/N) addition of 100 mM K + together with their PAGE assays of marker bands of HT24 (T 2 AG 3 ) 4 , HT48 (T 2 AG 3 ) 8 , and HT96 (T 2 AG 3 ) 16 and each sequence after 1 h and O/N addition of 100 mM K + for GTERT-d(FN), chl1 -d(FN), and PIG4-d(FN). The same DNA concentration of 100 μM was used in the experiments of CD, NMR and PAGE of this work.
    Figure Legend Snippet: The Effect on G4 formation of native G-rich sequences without flanking nucleotides. CD and NMR spectra of GTERT-d(FN), PIG4-d(FN), and chl1 -d(FN) in 10 mM Tris and after 1 h and overnight (O/N) addition of 100 mM K + together with their PAGE assays of marker bands of HT24 (T 2 AG 3 ) 4 , HT48 (T 2 AG 3 ) 8 , and HT96 (T 2 AG 3 ) 16 and each sequence after 1 h and O/N addition of 100 mM K + for GTERT-d(FN), chl1 -d(FN), and PIG4-d(FN). The same DNA concentration of 100 μM was used in the experiments of CD, NMR and PAGE of this work.

    Techniques Used: Nuclear Magnetic Resonance, Polyacrylamide Gel Electrophoresis, Marker, Sequencing, Concentration Assay

    The effect of middle loop base on G4 formation of (G 3 TG 3 N 4 G 3 TG 3 ) sequences. CD and NMR spectra of T4T-1, T4T-2, and T4T-3 in 10 mM Tris and after 1 h and overnight (O/N) addition of 100 mM K + together with their PAGE assays of marker bands of HT24 (T 2 AG 3 ) 4 , HT48 (T 2 AG 3 ) 8 , and HT96 (T 2 AG 3 ) 16 (lane 1) and each sequence after 1 h and O/N addition of 100 mM K + for T4T, T4T-1, T4T-2, and T4T-3. The same DNA concentration of 100 μM was used in the experiments of CD, NMR and PAGE of this work.
    Figure Legend Snippet: The effect of middle loop base on G4 formation of (G 3 TG 3 N 4 G 3 TG 3 ) sequences. CD and NMR spectra of T4T-1, T4T-2, and T4T-3 in 10 mM Tris and after 1 h and overnight (O/N) addition of 100 mM K + together with their PAGE assays of marker bands of HT24 (T 2 AG 3 ) 4 , HT48 (T 2 AG 3 ) 8 , and HT96 (T 2 AG 3 ) 16 (lane 1) and each sequence after 1 h and O/N addition of 100 mM K + for T4T, T4T-1, T4T-2, and T4T-3. The same DNA concentration of 100 μM was used in the experiments of CD, NMR and PAGE of this work.

    Techniques Used: Nuclear Magnetic Resonance, Polyacrylamide Gel Electrophoresis, Marker, Sequencing, Concentration Assay

    The effect of flanking nucleotides on G4 formation of (G 3 TG 3 N m G 3 TG 3 )-FN sequences. CD and NMR spectra of T16T-FN, T10T-FN, T4T-FN, T2T-FN, and TTT-FN in 10 mM Tris and after 1 h and overnight (O/N) addition of 100 mM K + together with their PAGE assays of marker bands of HT24 (T 2 AG 3 ) 4 , HT48 (T 2 AG 3 ) 8 , and HT96 (T 2 AG 3 ) 16 and each sequence after 1 h and O/N addition of 100 mM K + for T16T-FN, T10T-FN, T4T-FN, T2T-FN, and TTT-FN. The same DNA concentration of 100 μM was used in the experiments of CD, NMR and PAGE of this work.
    Figure Legend Snippet: The effect of flanking nucleotides on G4 formation of (G 3 TG 3 N m G 3 TG 3 )-FN sequences. CD and NMR spectra of T16T-FN, T10T-FN, T4T-FN, T2T-FN, and TTT-FN in 10 mM Tris and after 1 h and overnight (O/N) addition of 100 mM K + together with their PAGE assays of marker bands of HT24 (T 2 AG 3 ) 4 , HT48 (T 2 AG 3 ) 8 , and HT96 (T 2 AG 3 ) 16 and each sequence after 1 h and O/N addition of 100 mM K + for T16T-FN, T10T-FN, T4T-FN, T2T-FN, and TTT-FN. The same DNA concentration of 100 μM was used in the experiments of CD, NMR and PAGE of this work.

    Techniques Used: Nuclear Magnetic Resonance, Polyacrylamide Gel Electrophoresis, Marker, Sequencing, Concentration Assay

    The [K + ] dependent study on the effect of flanking nucleotides on G4 formation of mt10251-FN (mt10248). Time-dependent CD and NMR spectra of mt10248 in 10 mM Tris and after the addition of 20 mM K + ( a ) and 100 mM K + ( b ). The plots of arising CD signals at 265 nm of mt10248 fitted with single exponential after the addition of 20 mM K + and 100 mM K + ( c ). PAGE assays of marker bands of HT24 (T 2 AG 3 ) 4 , HT48 (T 2 AG 3 ) 8 , and HT96 (T 2 AG 3 ) 16 and mt10248 after 1h and O/N addition of 20 mM K + and 100 mM K + ( d ). The same DNA concentration of 100 μM was used in the experiments of CD, NMR and PAGE of this work.
    Figure Legend Snippet: The [K + ] dependent study on the effect of flanking nucleotides on G4 formation of mt10251-FN (mt10248). Time-dependent CD and NMR spectra of mt10248 in 10 mM Tris and after the addition of 20 mM K + ( a ) and 100 mM K + ( b ). The plots of arising CD signals at 265 nm of mt10248 fitted with single exponential after the addition of 20 mM K + and 100 mM K + ( c ). PAGE assays of marker bands of HT24 (T 2 AG 3 ) 4 , HT48 (T 2 AG 3 ) 8 , and HT96 (T 2 AG 3 ) 16 and mt10248 after 1h and O/N addition of 20 mM K + and 100 mM K + ( d ). The same DNA concentration of 100 μM was used in the experiments of CD, NMR and PAGE of this work.

    Techniques Used: Nuclear Magnetic Resonance, Polyacrylamide Gel Electrophoresis, Marker, Concentration Assay

    32) Product Images from "Changes in Human Erythrocyte Exposed to Organophosphate Flame Retardants: Tris(2-chloroethyl) Phosphate and Tris(1-chloro-2-propyl) Phosphate"

    Article Title: Changes in Human Erythrocyte Exposed to Organophosphate Flame Retardants: Tris(2-chloroethyl) Phosphate and Tris(1-chloro-2-propyl) Phosphate

    Journal: Materials

    doi: 10.3390/ma14133675

    Chemical formulas of tris(2-chloroethyl) phosphate (TCEP) and tris(1-chloro-2-propyl) phosphate (TCPP).
    Figure Legend Snippet: Chemical formulas of tris(2-chloroethyl) phosphate (TCEP) and tris(1-chloro-2-propyl) phosphate (TCPP).

    Techniques Used:

    33) Product Images from "Mechanism of Long-Chain Free Fatty Acid Protonation at the Membrane-Water Interface"

    Article Title: Mechanism of Long-Chain Free Fatty Acid Protonation at the Membrane-Water Interface

    Journal: Biophysical Journal

    doi: 10.1016/j.bpj.2018.04.011

    Dependence of pK a values of unsaturated FFAs on the number of double bonds. Liposomes were made from DOPC reconstituted with ( A ) arachidic (20:0), eicosenoic (20:1), eicosadienoic (20:2), and arachidonic (20:4) acids or ( B ) stearic (C18:0), oleic (C18:1), or γ -linolenic (C18:3) acids. The buffer solution contained 20 mM Na 2 SO 4 , 10 mM Tris, 10 mM MES, and 10 mM β -alanine. pK a, a and pK a are the apparent and intrinsic pK values of the corresponding FFAs. To see this figure in color, go online.
    Figure Legend Snippet: Dependence of pK a values of unsaturated FFAs on the number of double bonds. Liposomes were made from DOPC reconstituted with ( A ) arachidic (20:0), eicosenoic (20:1), eicosadienoic (20:2), and arachidonic (20:4) acids or ( B ) stearic (C18:0), oleic (C18:1), or γ -linolenic (C18:3) acids. The buffer solution contained 20 mM Na 2 SO 4 , 10 mM Tris, 10 mM MES, and 10 mM β -alanine. pK a, a and pK a are the apparent and intrinsic pK values of the corresponding FFAs. To see this figure in color, go online.

    Techniques Used:

    Surface potential and pK a values of saturated free fatty acids. ( A ) Dependence is shown of the liposome’s surface potential, Φ s , on buffer pH. Liposomes were either made from pure DOPC ( black spheres ) or from DOPC reconstituted with palmitic acid ( black squares ), or stearic acid ( black uptriangles ), or arachidic acid ( black downtriangles ). ( B ) Dependence is shown of pK a on the FFA chain length. The buffer solution contained 20 mM Na 2 SO 4 , 10 mM Tris, 10 mM MES, and 10 mM β -alanine. pK a, a and pK a are the apparent and intrinsic pK values of the corresponding FFAs. To see this figure in color, go online.
    Figure Legend Snippet: Surface potential and pK a values of saturated free fatty acids. ( A ) Dependence is shown of the liposome’s surface potential, Φ s , on buffer pH. Liposomes were either made from pure DOPC ( black spheres ) or from DOPC reconstituted with palmitic acid ( black squares ), or stearic acid ( black uptriangles ), or arachidic acid ( black downtriangles ). ( B ) Dependence is shown of pK a on the FFA chain length. The buffer solution contained 20 mM Na 2 SO 4 , 10 mM Tris, 10 mM MES, and 10 mM β -alanine. pK a, a and pK a are the apparent and intrinsic pK values of the corresponding FFAs. To see this figure in color, go online.

    Techniques Used:

    34) 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

    35) Product Images from "The N-Terminal Domain of PA from Bat-Derived Influenza-Like Virus H17N10 Has Endonuclease Activity"

    Article Title: The N-Terminal Domain of PA from Bat-Derived Influenza-Like Virus H17N10 Has Endonuclease Activity

    Journal: Journal of Virology

    doi: 10.1128/JVI.03270-13

    PAn from H17N10 has endonuclease activity. (A) Single-stranded DNA plasmid M13mp18 (50 ng/μl) was incubated with 20 μM wild-type H17N10 PAn for 1 h at 37°C in a buffer consisting of 20 mM Tris (pH 8) and 50 mM NaCl in the absence
    Figure Legend Snippet: PAn from H17N10 has endonuclease activity. (A) Single-stranded DNA plasmid M13mp18 (50 ng/μl) was incubated with 20 μM wild-type H17N10 PAn for 1 h at 37°C in a buffer consisting of 20 mM Tris (pH 8) and 50 mM NaCl in the absence

    Techniques Used: Activity Assay, Plasmid Preparation, Incubation

    36) Product Images from "Enhanced antitumor activity of surface-modified iron oxide nanoparticles and an α-tocopherol derivative in a rat model of mammary gland carcinosarcoma"

    Article Title: Enhanced antitumor activity of surface-modified iron oxide nanoparticles and an α-tocopherol derivative in a rat model of mammary gland carcinosarcoma

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S137574

    In vitro Fe 2+ release from ( A ) CuFe 2 O 4 , ( B ) γ-Fe 2 O 3 , and ( C ) γ-Fe 2 O 3 @PDMA nanoparticles in the presence of different concentrations of (♦) Toc and (■) Toc-6-OH. Nanoparticles (250 µg/mL) were incubated in a 0.9% NaCl solution and 10 mM Tris (pH 7.4) at 37°C for 24 h. Data are presented as the mean ± SE (n=5–8). *Significantly different from the absence of Toc and Toc-6-OH. # Significantly different from Toc.
    Figure Legend Snippet: In vitro Fe 2+ release from ( A ) CuFe 2 O 4 , ( B ) γ-Fe 2 O 3 , and ( C ) γ-Fe 2 O 3 @PDMA nanoparticles in the presence of different concentrations of (♦) Toc and (■) Toc-6-OH. Nanoparticles (250 µg/mL) were incubated in a 0.9% NaCl solution and 10 mM Tris (pH 7.4) at 37°C for 24 h. Data are presented as the mean ± SE (n=5–8). *Significantly different from the absence of Toc and Toc-6-OH. # Significantly different from Toc.

    Techniques Used: In Vitro, Incubation

    37) Product Images from "Identification and Toxicity Prediction of Biotransformation Molecules of Organophosphate Flame Retardants by Microbial Reactions in a Wastewater Treatment Plant"

    Article Title: Identification and Toxicity Prediction of Biotransformation Molecules of Organophosphate Flame Retardants by Microbial Reactions in a Wastewater Treatment Plant

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms22105376

    MS/MS fragments at oxidative biotransformation products of organophosphate flame retardants generated in biological reactor from wastewater treatment plant. ( a ) TB_414: biotransformation product (BTP) of tris (2-butoxyethyl) phosphate, ( b ) TC_290: BTP of tris (1-chloro-2-propyl) phosphate, ( c ) TP_342: BTP of triphenyl phosphate.
    Figure Legend Snippet: MS/MS fragments at oxidative biotransformation products of organophosphate flame retardants generated in biological reactor from wastewater treatment plant. ( a ) TB_414: biotransformation product (BTP) of tris (2-butoxyethyl) phosphate, ( b ) TC_290: BTP of tris (1-chloro-2-propyl) phosphate, ( c ) TP_342: BTP of triphenyl phosphate.

    Techniques Used: Tandem Mass Spectroscopy, Generated

    Biotransformation pathway and predicted toxicity of ( a ) tris (2-butoxyethyl) phosphate, ( b ) tris (1-chloro-2-propyl) phosphate, ( c ) triphenyl phosphate. The arrow represents the pathway to the formation of biotransformation products. The number of blocks indicates the estimated magnitude of the toxicity of substances.
    Figure Legend Snippet: Biotransformation pathway and predicted toxicity of ( a ) tris (2-butoxyethyl) phosphate, ( b ) tris (1-chloro-2-propyl) phosphate, ( c ) triphenyl phosphate. The arrow represents the pathway to the formation of biotransformation products. The number of blocks indicates the estimated magnitude of the toxicity of substances.

    Techniques Used:

    38) Product Images from "The N-terminal loop of IRAK-4 death domain regulates ordered assembly of the Myddosome signalling scaffold"

    Article Title: The N-terminal loop of IRAK-4 death domain regulates ordered assembly of the Myddosome signalling scaffold

    Journal: Scientific Reports

    doi: 10.1038/srep37267

    Phosphorylated Ser8 IRAK-4 death domain interferes with the Myddosome formation. ( A–D ) Absorbance Elution spectrum of a TSKgel SuperSW3000 analytical size exclusion column at 280 nm. ( A ) 0.5 mg.ml −1 of IRAK-4 death domain only. ( B ) 0.5 mg.ml −1 of MyD88 DD only. ( C ) 1:1 mix of MyD88 and non-phosphorylated IRAK-4 death domains concentrated to 0.5 mg.ml −1 prior to loading. ( D ) 1:1 mix of MyD88 and Ser8 phosphorylated IRAK-4 death domains concentrated to 0.5 mg.ml −1 prior loading. ( E ) Calibration of gel filtration using protein standard markers ( F ) 4–20% reducing Tris-Glycine SDS PAGE of fractions corresponding to peak 1, 2 and 3 of figure 15a. Fractions corresponding to peak 1 were concentrated using a 3 kDa MW cut-off Amicon Spin Filters for better visualisation of samples. One of three repeats is shown.
    Figure Legend Snippet: Phosphorylated Ser8 IRAK-4 death domain interferes with the Myddosome formation. ( A–D ) Absorbance Elution spectrum of a TSKgel SuperSW3000 analytical size exclusion column at 280 nm. ( A ) 0.5 mg.ml −1 of IRAK-4 death domain only. ( B ) 0.5 mg.ml −1 of MyD88 DD only. ( C ) 1:1 mix of MyD88 and non-phosphorylated IRAK-4 death domains concentrated to 0.5 mg.ml −1 prior to loading. ( D ) 1:1 mix of MyD88 and Ser8 phosphorylated IRAK-4 death domains concentrated to 0.5 mg.ml −1 prior loading. ( E ) Calibration of gel filtration using protein standard markers ( F ) 4–20% reducing Tris-Glycine SDS PAGE of fractions corresponding to peak 1, 2 and 3 of figure 15a. Fractions corresponding to peak 1 were concentrated using a 3 kDa MW cut-off Amicon Spin Filters for better visualisation of samples. One of three repeats is shown.

    Techniques Used: Filtration, SDS Page

    Reciprocal pull down assays confirm inability of Ser8-P death domain to interact with MyD88. ( A ) 4–20% reducing Tris-Glycine SDS PAGE of samples precipitated using a rabbit polyclonal anti-human MyD88 death domain antibody. Lane 1 to 3 are control lanes each loaded with 3 μg of the protein samples: lane 1-MyD88 DD, lane 2-non-phosphorylated IRAK-4 death domain, lane 3-Ser8 phosphorylated IRAK-4 death domain. Lanes 4 to 6 were loaded with the pull down experiment samples as indicated. ( B ) 4–20% reducing Tris-Glycine SDS PAGE of samples precipitated using a rabbit monoclonal anti-human IRAK-4 death domain antibody. Lane 1 to 3 were control lanes each loaded with 3 μg of the protein samples: lane 1 corresponded to MyD88 DD, lane 2-non-phosphorylated IRAK-4 death domain, lane 3-Ser8 phosphorylated IRAK-4 death domain. Lanes 4 to 7 were loaded with the pull down experiment samples as indicated. One repeat of three biological replicates is shown.
    Figure Legend Snippet: Reciprocal pull down assays confirm inability of Ser8-P death domain to interact with MyD88. ( A ) 4–20% reducing Tris-Glycine SDS PAGE of samples precipitated using a rabbit polyclonal anti-human MyD88 death domain antibody. Lane 1 to 3 are control lanes each loaded with 3 μg of the protein samples: lane 1-MyD88 DD, lane 2-non-phosphorylated IRAK-4 death domain, lane 3-Ser8 phosphorylated IRAK-4 death domain. Lanes 4 to 6 were loaded with the pull down experiment samples as indicated. ( B ) 4–20% reducing Tris-Glycine SDS PAGE of samples precipitated using a rabbit monoclonal anti-human IRAK-4 death domain antibody. Lane 1 to 3 were control lanes each loaded with 3 μg of the protein samples: lane 1 corresponded to MyD88 DD, lane 2-non-phosphorylated IRAK-4 death domain, lane 3-Ser8 phosphorylated IRAK-4 death domain. Lanes 4 to 7 were loaded with the pull down experiment samples as indicated. One repeat of three biological replicates is shown.

    Techniques Used: SDS Page

    Related Articles

    Plasmid Purification:

    Article Title: The role of irradiance and C-use strategies in tropical macroalgae photosynthetic response to ocean acidification
    Article Snippet: .. Inhibitors included acetazolamide (AZ, Sigma Aldrich) that blocks the dehydration of HCO3 − into CO2 via external carbonate anhydrase (CAext ) , pyridoxal (5) phosphate (PLP, Fisher Scientific) that inhibits active uptake of HCO3 − , Tris buffer (Trizma R, Sigma Aldrich) that interferes with proton pump acidification of the thalli boundary layer and sodium orthovanadate (vanadate, Sigma Aldrich) that obstructs plasmalemma ATPase H+ pumps . .. Solutions of AZ (200 μM), PLP (480 μM) and Tris (50 mM) were dissolved in filtered seawater (0.45 μm) followed by pH adjustment to 8.1 (1 M HCl).

    Derivative Assay:

    Article Title: Molecular Analysis of Pseudomonas aeruginosa: Epidemiological Investigation of Mastitis Outbreaks in Irish Dairy Herds †
    Article Snippet: Hybridization, washing, and color development of the membrane were carried out as described by Popovic et al. ( ). .. All PCRs were performed in final volumes of 50 μl each, containing 300 ng of genomic DNA, 100 pmol of RW3A primer (derived from a repetitive element in the genome of Mycoplasma pneumoniae [ ]), 5 μl of 10× PCR buffer (100 mM Tris-HCl [pH 9.0], 500 mM KCl, 1% Triton X-100), 8 μl of deoxynucleotide triphosphate mix (consisting of 1.25 mM [each] dATP, dCTP, dGTP, and dTTP), 2.5 mM MgCl2 , 0.5% dimethyl sulfoxide (Sigma), and 2.5 U of Taq DNA polymerase (Sigma). .. The RW3A primer (5′-TCG CTC AAA ACA ACG ACA CC-3′) ( ) was synthesized and polyacrylamide gel purified by Eurogentec (Abingdon, United Kingdom).

    Polymerase Chain Reaction:

    Article Title: Molecular Analysis of Pseudomonas aeruginosa: Epidemiological Investigation of Mastitis Outbreaks in Irish Dairy Herds †
    Article Snippet: Hybridization, washing, and color development of the membrane were carried out as described by Popovic et al. ( ). .. All PCRs were performed in final volumes of 50 μl each, containing 300 ng of genomic DNA, 100 pmol of RW3A primer (derived from a repetitive element in the genome of Mycoplasma pneumoniae [ ]), 5 μl of 10× PCR buffer (100 mM Tris-HCl [pH 9.0], 500 mM KCl, 1% Triton X-100), 8 μl of deoxynucleotide triphosphate mix (consisting of 1.25 mM [each] dATP, dCTP, dGTP, and dTTP), 2.5 mM MgCl2 , 0.5% dimethyl sulfoxide (Sigma), and 2.5 U of Taq DNA polymerase (Sigma). .. The RW3A primer (5′-TCG CTC AAA ACA ACG ACA CC-3′) ( ) was synthesized and polyacrylamide gel purified by Eurogentec (Abingdon, United Kingdom).

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    ( a ) Concentration-dependent SERS spectra using AgNPs and <t>Tris-EDTA</t> in the range of 0.005–10 μM of Cr(III); ( b ) A magnified view of the vibrational bands between 200 and 750 cm −1 in the Cr(III) concentration-dependent SERS spectra; ( c ) A calibration curve of the vibrational band intensities at ~563 cm −1 with respect to those at ~918 cm −1 . The inset shows a linear fitting result for the 0.05 and 1.0 μM regions.
    Tris Edta, 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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    Immunochemistry of whole-cell extracts with anti-(1→5)-α-arabinan probe, LM6. A, Immunoblotting on nitrocellulose with LM6 of material separated by SDS-PAGE. Material (10 μg of protein per lane) from WT MG fruit (lanes 1–3) and Cnr MG fruit (lanes 4–6) were prepared in sample buffer immediately (lanes 1 and 4) or after incubation in <t>Tris-buffer</t> (lanes 2 and 5) or incubation in Tris-buffer plus <t>Pronase</t> E (lanes 3 and 6). Significant amounts of LM6-reactive, Pronase E-sensitive material entered the gel from Cnr , but not WT material. R indicates top of resolving gel. M shows M r markers. B, Immunodot assay on nitrocellulose of material (0.4 μg of protein per dot) analyzed in A showing abundance of LM6 epitope in all samples.
    M Tris Hcl, 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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    Detection of bacteria induces changes in mitochondrial ETC complex activities and influences mitochondrial respiration and glycolysis. ( a ) Spectrophotometric activities of the indicated mitochondrial respiratory complexes, normalized to citrate synthase (CS) activity in mitochondria isolated from BMDMs treated or not with E. coli for 1.5h. Specific activities (in IU/mg protein) corresponding to 100% activity are 0.105±0.013, 0.040±0.008, 0.055±0.01, 0.1±0.03, 0.23±0.01, 0.02±0.003, 0.3±0.01 for CI, CII, CIII, CIV, CI+III, CII+III and CS, respectively. ( b ) Effect of EC-stimulation on WT BMDMs glutamate+malate-driven <t>ATP</t> synthesis. 100% activity corresponds to a rate of 153±24.9 nmol ATP/min/mg protein. ( c-f ) Oxygen consumption rate (OCR) upon sequential treatment of oligomycin (olig.), <t>CCCP,</t> and rotenone+antimycin (Rot.+Ant.) ( c ) spare respiratory capacity (SRC) ( d ), basal extracellular acidification rate (ECAR) ( e ) and extracellular lactate concentration ( f ) in WT BMDMs treated or not with EC for 2h. NS, not significant; *P
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    ( a ) Concentration-dependent SERS spectra using AgNPs and Tris-EDTA in the range of 0.005–10 μM of Cr(III); ( b ) A magnified view of the vibrational bands between 200 and 750 cm −1 in the Cr(III) concentration-dependent SERS spectra; ( c ) A calibration curve of the vibrational band intensities at ~563 cm −1 with respect to those at ~918 cm −1 . The inset shows a linear fitting result for the 0.05 and 1.0 μM regions.

    Journal: Sensors (Basel, Switzerland)

    Article Title: Silver Nanoparticle-Enhanced Resonance Raman Sensor of Chromium(III) in Seawater Samples

    doi: 10.3390/s150510088

    Figure Lengend Snippet: ( a ) Concentration-dependent SERS spectra using AgNPs and Tris-EDTA in the range of 0.005–10 μM of Cr(III); ( b ) A magnified view of the vibrational bands between 200 and 750 cm −1 in the Cr(III) concentration-dependent SERS spectra; ( c ) A calibration curve of the vibrational band intensities at ~563 cm −1 with respect to those at ~918 cm −1 . The inset shows a linear fitting result for the 0.05 and 1.0 μM regions.

    Article Snippet: Chemicals Cr(III) and the other ionic substances of NaCl, KNO3 , Mg(NO3 )2 , Ca(NO3 )2 , Cr(NO3 )3 , Mn(NO3 )2 , FeC2 O4 , Fe(NO3 )3 , Co(NO3 )2 , Ni(NO3 )2 , Cu(NO3 )2 , Zn(NO3 )2 , NH4 NO3 , Cd(NO3 )2 , Hg(NO3 )2 , Pb(NO3 )2 , and K2 Cr2 O7 (or Na2 CrO4 ) along with silver nitrate, sodium citrate, Tris-EDTA (pH 8.0), and EDTA acetic acid were purchased from Sigma Aldrich (St. Louis, MO, USA).

    Techniques: Concentration Assay

    ( a ) Surface-enhanced Raman spectroscopy (SERS) spectra of detection of Cr(III) using Tris(hydroxymethyl)aminomethane (Tris)-EDTA on AgNPs. Selective tests were performed for 50 μM of Cr 3+ , K + , Cd 2+ , Mg 2+ , Ca 2+ , Mn 2+ , Co 2+ , Na + , Cu 2+ , NH 4 + , Hg 2+ , Ni 2+ , Fe 3+ , Pb 2+ , Fe 2+ , and Zn 2+ ions; ( b ) Stick diagram of the intensities at ~563 cm −1 with respect to those at ~918 cm −1 versus 16 ionic species; ( c ) SERS spectra of the mixture of the 15 ions to test the competitive reactions; ( d ) Comparative SERS spectra of EDTA and EDTA-Cr 3+ on AgNPs at pH 4.0 and pH 8.0.

    Journal: Sensors (Basel, Switzerland)

    Article Title: Silver Nanoparticle-Enhanced Resonance Raman Sensor of Chromium(III) in Seawater Samples

    doi: 10.3390/s150510088

    Figure Lengend Snippet: ( a ) Surface-enhanced Raman spectroscopy (SERS) spectra of detection of Cr(III) using Tris(hydroxymethyl)aminomethane (Tris)-EDTA on AgNPs. Selective tests were performed for 50 μM of Cr 3+ , K + , Cd 2+ , Mg 2+ , Ca 2+ , Mn 2+ , Co 2+ , Na + , Cu 2+ , NH 4 + , Hg 2+ , Ni 2+ , Fe 3+ , Pb 2+ , Fe 2+ , and Zn 2+ ions; ( b ) Stick diagram of the intensities at ~563 cm −1 with respect to those at ~918 cm −1 versus 16 ionic species; ( c ) SERS spectra of the mixture of the 15 ions to test the competitive reactions; ( d ) Comparative SERS spectra of EDTA and EDTA-Cr 3+ on AgNPs at pH 4.0 and pH 8.0.

    Article Snippet: Chemicals Cr(III) and the other ionic substances of NaCl, KNO3 , Mg(NO3 )2 , Ca(NO3 )2 , Cr(NO3 )3 , Mn(NO3 )2 , FeC2 O4 , Fe(NO3 )3 , Co(NO3 )2 , Ni(NO3 )2 , Cu(NO3 )2 , Zn(NO3 )2 , NH4 NO3 , Cd(NO3 )2 , Hg(NO3 )2 , Pb(NO3 )2 , and K2 Cr2 O7 (or Na2 CrO4 ) along with silver nitrate, sodium citrate, Tris-EDTA (pH 8.0), and EDTA acetic acid were purchased from Sigma Aldrich (St. Louis, MO, USA).

    Techniques: Raman Spectroscopy

    Immunochemistry of whole-cell extracts with anti-(1→5)-α-arabinan probe, LM6. A, Immunoblotting on nitrocellulose with LM6 of material separated by SDS-PAGE. Material (10 μg of protein per lane) from WT MG fruit (lanes 1–3) and Cnr MG fruit (lanes 4–6) were prepared in sample buffer immediately (lanes 1 and 4) or after incubation in Tris-buffer (lanes 2 and 5) or incubation in Tris-buffer plus Pronase E (lanes 3 and 6). Significant amounts of LM6-reactive, Pronase E-sensitive material entered the gel from Cnr , but not WT material. R indicates top of resolving gel. M shows M r markers. B, Immunodot assay on nitrocellulose of material (0.4 μg of protein per dot) analyzed in A showing abundance of LM6 epitope in all samples.

    Journal: Plant Physiology

    Article Title: Altered Middle Lamella Homogalacturonan and Disrupted Deposition of (1- > 5)-?-l-Arabinan in the Pericarp of Cnr, a Ripening Mutant of Tomato 1

    doi:

    Figure Lengend Snippet: Immunochemistry of whole-cell extracts with anti-(1→5)-α-arabinan probe, LM6. A, Immunoblotting on nitrocellulose with LM6 of material separated by SDS-PAGE. Material (10 μg of protein per lane) from WT MG fruit (lanes 1–3) and Cnr MG fruit (lanes 4–6) were prepared in sample buffer immediately (lanes 1 and 4) or after incubation in Tris-buffer (lanes 2 and 5) or incubation in Tris-buffer plus Pronase E (lanes 3 and 6). Significant amounts of LM6-reactive, Pronase E-sensitive material entered the gel from Cnr , but not WT material. R indicates top of resolving gel. M shows M r markers. B, Immunodot assay on nitrocellulose of material (0.4 μg of protein per dot) analyzed in A showing abundance of LM6 epitope in all samples.

    Article Snippet: The powder (2 mL) was suspended in 50 m m Tris-HCl, pH 6.5, or 50 m m Tris-HCl, pH 6.5, containing Pronase E (Sigma) at 1 mg mL−1 .

    Techniques: SDS Page, Incubation

    Detection of bacteria induces changes in mitochondrial ETC complex activities and influences mitochondrial respiration and glycolysis. ( a ) Spectrophotometric activities of the indicated mitochondrial respiratory complexes, normalized to citrate synthase (CS) activity in mitochondria isolated from BMDMs treated or not with E. coli for 1.5h. Specific activities (in IU/mg protein) corresponding to 100% activity are 0.105±0.013, 0.040±0.008, 0.055±0.01, 0.1±0.03, 0.23±0.01, 0.02±0.003, 0.3±0.01 for CI, CII, CIII, CIV, CI+III, CII+III and CS, respectively. ( b ) Effect of EC-stimulation on WT BMDMs glutamate+malate-driven ATP synthesis. 100% activity corresponds to a rate of 153±24.9 nmol ATP/min/mg protein. ( c-f ) Oxygen consumption rate (OCR) upon sequential treatment of oligomycin (olig.), CCCP, and rotenone+antimycin (Rot.+Ant.) ( c ) spare respiratory capacity (SRC) ( d ), basal extracellular acidification rate (ECAR) ( e ) and extracellular lactate concentration ( f ) in WT BMDMs treated or not with EC for 2h. NS, not significant; *P

    Journal: Nature immunology

    Article Title: Mitochondrial respiratory chain adaptations in macrophages contribute to antibacterial host defence

    doi: 10.1038/ni.3509

    Figure Lengend Snippet: Detection of bacteria induces changes in mitochondrial ETC complex activities and influences mitochondrial respiration and glycolysis. ( a ) Spectrophotometric activities of the indicated mitochondrial respiratory complexes, normalized to citrate synthase (CS) activity in mitochondria isolated from BMDMs treated or not with E. coli for 1.5h. Specific activities (in IU/mg protein) corresponding to 100% activity are 0.105±0.013, 0.040±0.008, 0.055±0.01, 0.1±0.03, 0.23±0.01, 0.02±0.003, 0.3±0.01 for CI, CII, CIII, CIV, CI+III, CII+III and CS, respectively. ( b ) Effect of EC-stimulation on WT BMDMs glutamate+malate-driven ATP synthesis. 100% activity corresponds to a rate of 153±24.9 nmol ATP/min/mg protein. ( c-f ) Oxygen consumption rate (OCR) upon sequential treatment of oligomycin (olig.), CCCP, and rotenone+antimycin (Rot.+Ant.) ( c ) spare respiratory capacity (SRC) ( d ), basal extracellular acidification rate (ECAR) ( e ) and extracellular lactate concentration ( f ) in WT BMDMs treated or not with EC for 2h. NS, not significant; *P

    Article Snippet: 3-nitropropionic acid (NPA), succinate, succinate hexahydrate, glutamate, malate disodium-salt, fumarate, dimethyl-fumarate, dimethyl succinate, dimethyl malonate (DM), itaconic acid, thenoyltrifluoroacetone (TTFA), carbonilcyanide p-triflouromethoxyphenylhydrazone (FCCP), CCCP, oligomycin, rotenone, antimycin A, ubiquinone, sn-glycerol 3-phosphate, oxidized cytochrome c, adenosine tri-phosphate (ATP), adenosine di-phosphate (ADP), phenazine methosulfate (PMS) and digitonin were all from Sigma.

    Techniques: Activity Assay, Isolation, Concentration Assay