Structured Review

Merck KGaA na hepes
2–6 dithiopurine stabilizes human lysosomal alpha-galactosidase in thermal shift assay. Panel A. Fabrazyme ® (in <t>Na-Hepes</t> 20 mM, NaCl 150 mM, pH 7.4) was equilibrated in the presence of ligands dissolved in DMSO 20%: DGJ 1 microM (empty triangle) or 40 microM (empty circle); <t>DTP</t> 6 mM (empty diamond); DTP 6 mM plus DGJ 1 microM (filled circle); DTP 6 mM plus DGJ 40 microM (filled triangle). A control (with only DMSO) was also shown (x). Panel B. Fabrazyme ® (in Na-Hepes 20 mM, NaCl 150 mM, pH 7.4) was incubated in the presence of DTP 6 mM for 1 hour at 4°C then DTP was eliminated by dialysis. A control experiment was conducted by incubating the enzyme only with DMSO. Both the aliquots of Fabrazyme ® were analysed by thermal shift assay in the presence of DTP 6 mM or in the presence of DMSO. Filled squares: DTP/DTP; open squares: DMSO/DTP; filled circles: DTP/DMSO; open circles: DMSO/DMSO where the first word of the label corresponds to the pretreatment and the second part corresponds to the presence of the compound during the thermal scan. The protein samples were heated from 20 to 90° at 1°C/min in the presence of Sypro Orange 2.5x. Data were shown as normalized curves.
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Images

1) Product Images from "Identification of an Allosteric Binding Site on Human Lysosomal Alpha-Galactosidase Opens the Way to New Pharmacological Chaperones for Fabry Disease"

Article Title: Identification of an Allosteric Binding Site on Human Lysosomal Alpha-Galactosidase Opens the Way to New Pharmacological Chaperones for Fabry Disease

Journal: PLoS ONE

doi: 10.1371/journal.pone.0165463

2–6 dithiopurine stabilizes human lysosomal alpha-galactosidase in thermal shift assay. Panel A. Fabrazyme ® (in Na-Hepes 20 mM, NaCl 150 mM, pH 7.4) was equilibrated in the presence of ligands dissolved in DMSO 20%: DGJ 1 microM (empty triangle) or 40 microM (empty circle); DTP 6 mM (empty diamond); DTP 6 mM plus DGJ 1 microM (filled circle); DTP 6 mM plus DGJ 40 microM (filled triangle). A control (with only DMSO) was also shown (x). Panel B. Fabrazyme ® (in Na-Hepes 20 mM, NaCl 150 mM, pH 7.4) was incubated in the presence of DTP 6 mM for 1 hour at 4°C then DTP was eliminated by dialysis. A control experiment was conducted by incubating the enzyme only with DMSO. Both the aliquots of Fabrazyme ® were analysed by thermal shift assay in the presence of DTP 6 mM or in the presence of DMSO. Filled squares: DTP/DTP; open squares: DMSO/DTP; filled circles: DTP/DMSO; open circles: DMSO/DMSO where the first word of the label corresponds to the pretreatment and the second part corresponds to the presence of the compound during the thermal scan. The protein samples were heated from 20 to 90° at 1°C/min in the presence of Sypro Orange 2.5x. Data were shown as normalized curves.
Figure Legend Snippet: 2–6 dithiopurine stabilizes human lysosomal alpha-galactosidase in thermal shift assay. Panel A. Fabrazyme ® (in Na-Hepes 20 mM, NaCl 150 mM, pH 7.4) was equilibrated in the presence of ligands dissolved in DMSO 20%: DGJ 1 microM (empty triangle) or 40 microM (empty circle); DTP 6 mM (empty diamond); DTP 6 mM plus DGJ 1 microM (filled circle); DTP 6 mM plus DGJ 40 microM (filled triangle). A control (with only DMSO) was also shown (x). Panel B. Fabrazyme ® (in Na-Hepes 20 mM, NaCl 150 mM, pH 7.4) was incubated in the presence of DTP 6 mM for 1 hour at 4°C then DTP was eliminated by dialysis. A control experiment was conducted by incubating the enzyme only with DMSO. Both the aliquots of Fabrazyme ® were analysed by thermal shift assay in the presence of DTP 6 mM or in the presence of DMSO. Filled squares: DTP/DTP; open squares: DMSO/DTP; filled circles: DTP/DMSO; open circles: DMSO/DMSO where the first word of the label corresponds to the pretreatment and the second part corresponds to the presence of the compound during the thermal scan. The protein samples were heated from 20 to 90° at 1°C/min in the presence of Sypro Orange 2.5x. Data were shown as normalized curves.

Techniques Used: Thermal Shift Assay, Incubation

2) Product Images from "Solution Properties of Tetramethylrhodamine-Modified G-Actin"

Article Title: Solution Properties of Tetramethylrhodamine-Modified G-Actin

Journal: Biophysical Journal

doi:

Nucleotide exchange inhibition by gelsolin-S1. Nucleotide exchange in the gelsolin-S1 complex with Ca-ATP-actin ( A ) and Mg-ADP-actin ( B ) was monitored via changes in fluorescence intensity upon ɛ -nucleotide release from, or incorporation into, the nucleotide-binding cleft on actin. 100 μ M ɛ -ATP ( A ) or ɛ -ADP ( B ) was added at the zero time to 2.0 μ M actin in 10 mM HEPES buffer, at pH 7.5, containing 1 mM DTT, and either 30 μ M CaCl 2 and 5.0 μ M ATP in A , or 30 μ M MgCl 2 and 5.0 μ M ADP in B . Etheno-nucleotide release was initiated by the addition of 1.0 mM ATP (marked by arrowheads above curves a and b ). Curve a , data in gray; no gelsolin-S1 present. Curve b , data in black; 4.0 μ M gelsolin-S1 was added to this sample after the fluorescence increase due to etheno-nucleotide incorporation reached a plateau (indicated by the arrow). Curve c , data in dark gray; 4.0 μ M gelsolin-S1 was added 5 min before the addition of ɛ -ATP or ɛ -ADP to G-actin (at the zero time). In all cases nucleotide exchange was abolished in the presence of gelsolin-S1.
Figure Legend Snippet: Nucleotide exchange inhibition by gelsolin-S1. Nucleotide exchange in the gelsolin-S1 complex with Ca-ATP-actin ( A ) and Mg-ADP-actin ( B ) was monitored via changes in fluorescence intensity upon ɛ -nucleotide release from, or incorporation into, the nucleotide-binding cleft on actin. 100 μ M ɛ -ATP ( A ) or ɛ -ADP ( B ) was added at the zero time to 2.0 μ M actin in 10 mM HEPES buffer, at pH 7.5, containing 1 mM DTT, and either 30 μ M CaCl 2 and 5.0 μ M ATP in A , or 30 μ M MgCl 2 and 5.0 μ M ADP in B . Etheno-nucleotide release was initiated by the addition of 1.0 mM ATP (marked by arrowheads above curves a and b ). Curve a , data in gray; no gelsolin-S1 present. Curve b , data in black; 4.0 μ M gelsolin-S1 was added to this sample after the fluorescence increase due to etheno-nucleotide incorporation reached a plateau (indicated by the arrow). Curve c , data in dark gray; 4.0 μ M gelsolin-S1 was added 5 min before the addition of ɛ -ATP or ɛ -ADP to G-actin (at the zero time). In all cases nucleotide exchange was abolished in the presence of gelsolin-S1.

Techniques Used: Inhibition, Fluorescence, Binding Assay

3) Product Images from "Analysis of the substrate recognition state of TDP-43 to single-stranded DNA using fluorescence correlation spectroscopy"

Article Title: Analysis of the substrate recognition state of TDP-43 to single-stranded DNA using fluorescence correlation spectroscopy

Journal: Biochemistry and Biophysics Reports

doi: 10.1016/j.bbrep.2018.03.009

Autocorrelation functions (ACFs) of fluorescently labeled ssDNA in the presence and absence of purified TDP-43. Normalized ACFs of Alexa Fluor 647-labeled TG 12 (TG; A C) or T 20 (TT; B D) in 50 mM HEPES-KOH buffer without NP-40 are shown in the presence and absence of wild type TDP-43 (WT; 42.1 ng) or the A315T mutant (AT; 67.5 ng; magenta and green, respectively).
Figure Legend Snippet: Autocorrelation functions (ACFs) of fluorescently labeled ssDNA in the presence and absence of purified TDP-43. Normalized ACFs of Alexa Fluor 647-labeled TG 12 (TG; A C) or T 20 (TT; B D) in 50 mM HEPES-KOH buffer without NP-40 are shown in the presence and absence of wild type TDP-43 (WT; 42.1 ng) or the A315T mutant (AT; 67.5 ng; magenta and green, respectively).

Techniques Used: Labeling, Purification, Mutagenesis

4) Product Images from "Optimizing adipogenic transdifferentiation of bovine mesenchymal stem cells: a prominent role of ascorbic acid in FABP4 induction"

Article Title: Optimizing adipogenic transdifferentiation of bovine mesenchymal stem cells: a prominent role of ascorbic acid in FABP4 induction

Journal: Adipocyte

doi: 10.1080/21623945.2020.1720480

Relative mRNA expression for NT5E, THY1, ENG, PDGFRα, FABP4, LPL, GLUT4 and PPARγ in bovine subcutaneous ASC after in vitro transdifferentiation. Quantitative reverse transcription PCR analysis of NT5E (A), THY1 (B), ENG (C), PDGFRα (D), FABP4 (E), LPL (F), GLUT4 (G) and PPARγ (H) are given as the mean ± SEM of four animals with three replicates. Pre, pre-adipocyte (RNA extracted before induction); Con, negative control; TRT = treatment; FBS, foetal bovine serum; AsA, ascorbic acid; BSL, bovine serum lipids. Letters (a-e) are used to denote statistical difference; columns within one graph are different if they do not share a common letter ( P
Figure Legend Snippet: Relative mRNA expression for NT5E, THY1, ENG, PDGFRα, FABP4, LPL, GLUT4 and PPARγ in bovine subcutaneous ASC after in vitro transdifferentiation. Quantitative reverse transcription PCR analysis of NT5E (A), THY1 (B), ENG (C), PDGFRα (D), FABP4 (E), LPL (F), GLUT4 (G) and PPARγ (H) are given as the mean ± SEM of four animals with three replicates. Pre, pre-adipocyte (RNA extracted before induction); Con, negative control; TRT = treatment; FBS, foetal bovine serum; AsA, ascorbic acid; BSL, bovine serum lipids. Letters (a-e) are used to denote statistical difference; columns within one graph are different if they do not share a common letter ( P

Techniques Used: Expressing, In Vitro, Polymerase Chain Reaction, Negative Control

5) Product Images from "Diverse allosteric and catalytic functions of tetrameric d-lactate dehydrogenases from three Gram-negative bacteria"

Article Title: Diverse allosteric and catalytic functions of tetrameric d-lactate dehydrogenases from three Gram-negative bacteria

Journal: AMB Express

doi: 10.1186/s13568-014-0076-1

pH-stability of FNLDH (a), PALDH (b), and ECLDH (c), and heat-stability of the enzymes (d). a-c) Each enzyme was treated at 30°C for 1 h in sodium citrate (open circles), sodium acetate (closed circles), MES-NaOH (open triangles), MOPS-NaOH (closed triangles), HEPES-NaOH (open squares), Bicine-NaOH ( N,N -Bis(2-hydroxyethyl)glycine) (closed squares), CHES-NaOH ( N -cyclohexyl-2-aminoethanesulfonic acid) (open diamonds), and CAPS-NaOH ( N -cyclohexyl-3-aminopropanesulfonic acid) (closed diamonds) buffers. d) FNLDH (white circles and solid lines), PALDH (grey circles and dashed lines), and ECLDH (black circles and dotted lines) were treated at various temperatures for 30 min in the buffers described in ‘Materials and methods’.
Figure Legend Snippet: pH-stability of FNLDH (a), PALDH (b), and ECLDH (c), and heat-stability of the enzymes (d). a-c) Each enzyme was treated at 30°C for 1 h in sodium citrate (open circles), sodium acetate (closed circles), MES-NaOH (open triangles), MOPS-NaOH (closed triangles), HEPES-NaOH (open squares), Bicine-NaOH ( N,N -Bis(2-hydroxyethyl)glycine) (closed squares), CHES-NaOH ( N -cyclohexyl-2-aminoethanesulfonic acid) (open diamonds), and CAPS-NaOH ( N -cyclohexyl-3-aminopropanesulfonic acid) (closed diamonds) buffers. d) FNLDH (white circles and solid lines), PALDH (grey circles and dashed lines), and ECLDH (black circles and dotted lines) were treated at various temperatures for 30 min in the buffers described in ‘Materials and methods’.

Techniques Used:

pH dependence of kinetic parameters on pyruvate reduction. White, grey, and black circles indicate FNLDH, PALDH, and ECLDH, respectively. The k cat (a) , S 0.5 (b) , and k cat / S 0.5 data (c) are plotted logarithmically, and the Hill coefficient (d) is plotted linearly. The buffers used for the assay were sodium acetate buffer (pH 4.5, 5.0, and 5.5; circles), MES-NaOH buffer (pH 5.5, 6.0, and 6.5; triangles), MOPS-NaOH buffer (pH 6.5, 7.0, and 7.5; squares), HEPES-NaOH buffer (pH 7.0, 7.5 and 8.0; diamonds), and Bicine-NaOH buffer (pH 8.0, 8.5, and 9.0; hexagons). We adjusted the pH of each buffer solution prior to the addition of substrate and cofactor, and confirmed that the pH value is not affected even in the presence of high concentrations of substrate.
Figure Legend Snippet: pH dependence of kinetic parameters on pyruvate reduction. White, grey, and black circles indicate FNLDH, PALDH, and ECLDH, respectively. The k cat (a) , S 0.5 (b) , and k cat / S 0.5 data (c) are plotted logarithmically, and the Hill coefficient (d) is plotted linearly. The buffers used for the assay were sodium acetate buffer (pH 4.5, 5.0, and 5.5; circles), MES-NaOH buffer (pH 5.5, 6.0, and 6.5; triangles), MOPS-NaOH buffer (pH 6.5, 7.0, and 7.5; squares), HEPES-NaOH buffer (pH 7.0, 7.5 and 8.0; diamonds), and Bicine-NaOH buffer (pH 8.0, 8.5, and 9.0; hexagons). We adjusted the pH of each buffer solution prior to the addition of substrate and cofactor, and confirmed that the pH value is not affected even in the presence of high concentrations of substrate.

Techniques Used:

6) Product Images from "Broad spectrum antiviral activity for paramyxoviruses is modulated by biophysical properties of fusion inhibitory peptides"

Article Title: Broad spectrum antiviral activity for paramyxoviruses is modulated by biophysical properties of fusion inhibitory peptides

Journal: Scientific Reports

doi: 10.1038/srep43610

Spectroscopic characterization of VG peptides. ( a ) Environmental effects on the tryptophan residue of VG peptides. Normalized fluorescence excitation (dashed line) and emission (solid line) spectra of each peptide and Trp in HEPES buffer 10 mM pH 7.4 in NaCl 150 mM. ( b ) Concentration-dependent aggregation of the VG peptides, evaluated by ANS (12.8 μM) fluorescence intensity (λexc = 369 nm; emission spectra integrated from 400 to 600 nm). The values are means ± standart error of the mean (±s.e.m) of at least three experiments. Statistical analysis was performed using a 2-way ANOVA test (* P
Figure Legend Snippet: Spectroscopic characterization of VG peptides. ( a ) Environmental effects on the tryptophan residue of VG peptides. Normalized fluorescence excitation (dashed line) and emission (solid line) spectra of each peptide and Trp in HEPES buffer 10 mM pH 7.4 in NaCl 150 mM. ( b ) Concentration-dependent aggregation of the VG peptides, evaluated by ANS (12.8 μM) fluorescence intensity (λexc = 369 nm; emission spectra integrated from 400 to 600 nm). The values are means ± standart error of the mean (±s.e.m) of at least three experiments. Statistical analysis was performed using a 2-way ANOVA test (* P

Techniques Used: Fluorescence, Concentration Assay

7) Product Images from "Development of an oxygenation culture method for activating the liver-specific functions of HepG2 cells utilizing a collagen vitrigel membrane chamber"

Article Title: Development of an oxygenation culture method for activating the liver-specific functions of HepG2 cells utilizing a collagen vitrigel membrane chamber

Journal: Cytotechnology

doi: 10.1007/s10616-015-9934-1

Urea synthesis level of HepG2 cells at day 3 in each culture method. Closed, open, dotted, and hatched columns represent the liquid–liquid interface culture method using a control Millicell chamber with a 1.0 µm-porous PET membrane, the liquid–solid, liquid–liquid and liquid–gas interface culture methods using CVM chambers, respectively. Each value represents the mean ± SD (n = 3). * P
Figure Legend Snippet: Urea synthesis level of HepG2 cells at day 3 in each culture method. Closed, open, dotted, and hatched columns represent the liquid–liquid interface culture method using a control Millicell chamber with a 1.0 µm-porous PET membrane, the liquid–solid, liquid–liquid and liquid–gas interface culture methods using CVM chambers, respectively. Each value represents the mean ± SD (n = 3). * P

Techniques Used: Positron Emission Tomography

CYP3A4 activity level of HepG2 cells at day 3 in each culture method. Closed , open , dotted , and hatched columns represent the liquid–liquid interface culture method using a control Millicell chamber with a 1.0 µm-porous PET membrane, the liquid–solid, liquid–liquid and liquid–gas interface culture methods using CVM chambers, respectively. Each value represents the mean ± SD (n = 3). * P
Figure Legend Snippet: CYP3A4 activity level of HepG2 cells at day 3 in each culture method. Closed , open , dotted , and hatched columns represent the liquid–liquid interface culture method using a control Millicell chamber with a 1.0 µm-porous PET membrane, the liquid–solid, liquid–liquid and liquid–gas interface culture methods using CVM chambers, respectively. Each value represents the mean ± SD (n = 3). * P

Techniques Used: Activity Assay, Positron Emission Tomography

Fluorescence microscopic observation of FD-incorporated HepG2 cells at day 3 in each culture method. The cells were cultured on the liquid–liquid interface using a control Millicell chamber with a 1.0 µm-porous PET membrane ( a , e ), the liquid–solid ( b , f ), liquid–liquid ( c , g ) and liquid–gas ( d , h ) interfaces using CVM chambers. To assay the ADME of FD, the metabolized fluorescein at 10 min ( a – d ) and 60 min ( e – h ) after incorporating FD into the cells was observed under the same visual field in each culture system. Asterisks indicate the cells expressing the fluorescence in the interstices between the neighboring cells rather than inside the cells ( d ). Arrowheads indicate the excreted fluorescein into the bile canaliculus-like networks ( h ). Scale bars represent 50 µm
Figure Legend Snippet: Fluorescence microscopic observation of FD-incorporated HepG2 cells at day 3 in each culture method. The cells were cultured on the liquid–liquid interface using a control Millicell chamber with a 1.0 µm-porous PET membrane ( a , e ), the liquid–solid ( b , f ), liquid–liquid ( c , g ) and liquid–gas ( d , h ) interfaces using CVM chambers. To assay the ADME of FD, the metabolized fluorescein at 10 min ( a – d ) and 60 min ( e – h ) after incorporating FD into the cells was observed under the same visual field in each culture system. Asterisks indicate the cells expressing the fluorescence in the interstices between the neighboring cells rather than inside the cells ( d ). Arrowheads indicate the excreted fluorescein into the bile canaliculus-like networks ( h ). Scale bars represent 50 µm

Techniques Used: Fluorescence, Cell Culture, Positron Emission Tomography, Expressing

Schematic illustration of three different culture methods of HepG2 cells using culture chambers. “Liquid–solid interface” culture method ( a ), “liquid–liquid interface” culture method ( b ), and “liquid–gas interface” culture method ( c ). Thin black line with the sequence of “ ”-symbols, gray zone and white zone represent a culture chamber, a culture medium and a humidified gas of 5 % CO 2 in air, respectively. Here, the sequence of “ ”-symbols represents a membrane of culture chambers; CVM of CVM chambers or a porous PET membrane of control Millicell chambers. Also, thick black line in ( a ) represents a plastic culture dish with a diameter of 60 mm and that in ( b ) and ( c ) does a well of 12 well-plastic culture plate
Figure Legend Snippet: Schematic illustration of three different culture methods of HepG2 cells using culture chambers. “Liquid–solid interface” culture method ( a ), “liquid–liquid interface” culture method ( b ), and “liquid–gas interface” culture method ( c ). Thin black line with the sequence of “ ”-symbols, gray zone and white zone represent a culture chamber, a culture medium and a humidified gas of 5 % CO 2 in air, respectively. Here, the sequence of “ ”-symbols represents a membrane of culture chambers; CVM of CVM chambers or a porous PET membrane of control Millicell chambers. Also, thick black line in ( a ) represents a plastic culture dish with a diameter of 60 mm and that in ( b ) and ( c ) does a well of 12 well-plastic culture plate

Techniques Used: Sequencing, Positron Emission Tomography

Gross observation showing advantages of CVM chambers in comparison to control Millicell chambers. In order to design three different culture methods described in Fig. 1 using CVM chambers and their control Millicell chambers, 0.50 ml of culture medium was poured in the inside of each chamber pre-set on the solid ( a , d , g ), liquid ( b , e , h ), and gas ( c , f , i ) surfaces and the capability for preserving the inside culture medium was examined by keeping each condition for 2 h. The chambers in ( a – c ), ( d – f ), and ( g – i ) represent CVM chambers, control Millicell chambers with a 1.0 µm-porous PET membrane, and those with a 0.4 µm-porous PET membrane, respectively
Figure Legend Snippet: Gross observation showing advantages of CVM chambers in comparison to control Millicell chambers. In order to design three different culture methods described in Fig. 1 using CVM chambers and their control Millicell chambers, 0.50 ml of culture medium was poured in the inside of each chamber pre-set on the solid ( a , d , g ), liquid ( b , e , h ), and gas ( c , f , i ) surfaces and the capability for preserving the inside culture medium was examined by keeping each condition for 2 h. The chambers in ( a – c ), ( d – f ), and ( g – i ) represent CVM chambers, control Millicell chambers with a 1.0 µm-porous PET membrane, and those with a 0.4 µm-porous PET membrane, respectively

Techniques Used: Preserving, Positron Emission Tomography

Albumin secretion level of HepG2 cells at day 3 in each culture method. Closed , open , dotted , and hatched columns represent the liquid–liquid interface culture method using a control Millicell chamber with a 1.0 µm-porous PET membrane, the liquid–solid, liquid–liquid and liquid–gas interface culture methods using CVM chambers, respectively. Each value represents the mean ± SD (n = 3). * P
Figure Legend Snippet: Albumin secretion level of HepG2 cells at day 3 in each culture method. Closed , open , dotted , and hatched columns represent the liquid–liquid interface culture method using a control Millicell chamber with a 1.0 µm-porous PET membrane, the liquid–solid, liquid–liquid and liquid–gas interface culture methods using CVM chambers, respectively. Each value represents the mean ± SD (n = 3). * P

Techniques Used: Positron Emission Tomography

Microscopic observation of HepG2 cells cultured in control Millicell and CVM chambers for 3 days. HepG2 cells seeded in a control Millicell chamber with a 1.0 µm-porous PET membrane ( a , e ) were cultured on the liquid–liquid interface. Also, the cells seeded in CVM chambers pre-set on the solid surface were cultured on the liquid–solid interface for 48 h to induce the uniform cell growth and subsequently were cultured for 24 h on the liquid–solid ( b , f ), liquid–liquid ( c , g ) and liquid–gas interfaces ( d , h ). The same visual field of each culture system was observed with a phase-contrast microscope ( a – d ) and a fluorescent microscope ( e – h ) after staining the cells with calcein ( green ) and ethidium homodimer-1 ( red ). Arrowheads indicate typical bile canaliculus-like aspects ( d ). Scale bars represent 50 µm. (Color figure online)
Figure Legend Snippet: Microscopic observation of HepG2 cells cultured in control Millicell and CVM chambers for 3 days. HepG2 cells seeded in a control Millicell chamber with a 1.0 µm-porous PET membrane ( a , e ) were cultured on the liquid–liquid interface. Also, the cells seeded in CVM chambers pre-set on the solid surface were cultured on the liquid–solid interface for 48 h to induce the uniform cell growth and subsequently were cultured for 24 h on the liquid–solid ( b , f ), liquid–liquid ( c , g ) and liquid–gas interfaces ( d , h ). The same visual field of each culture system was observed with a phase-contrast microscope ( a – d ) and a fluorescent microscope ( e – h ) after staining the cells with calcein ( green ) and ethidium homodimer-1 ( red ). Arrowheads indicate typical bile canaliculus-like aspects ( d ). Scale bars represent 50 µm. (Color figure online)

Techniques Used: Cell Culture, Positron Emission Tomography, Microscopy, Staining

8) Product Images from "Analysis of the substrate recognition state of TDP-43 to single-stranded DNA using fluorescence correlation spectroscopy"

Article Title: Analysis of the substrate recognition state of TDP-43 to single-stranded DNA using fluorescence correlation spectroscopy

Journal: Biochemistry and Biophysics Reports

doi: 10.1016/j.bbrep.2018.03.009

Autocorrelation functions (ACFs) of fluorescently labeled ssDNA in the presence and absence of purified TDP-43. Normalized ACFs of Alexa Fluor 647-labeled TG 12 (TG; A C) or T 20 (TT; B D) in 50 mM HEPES-KOH buffer without NP-40 are shown in the presence and absence of wild type TDP-43 (WT; 42.1 ng) or the A315T mutant (AT; 67.5 ng; magenta and green, respectively).
Figure Legend Snippet: Autocorrelation functions (ACFs) of fluorescently labeled ssDNA in the presence and absence of purified TDP-43. Normalized ACFs of Alexa Fluor 647-labeled TG 12 (TG; A C) or T 20 (TT; B D) in 50 mM HEPES-KOH buffer without NP-40 are shown in the presence and absence of wild type TDP-43 (WT; 42.1 ng) or the A315T mutant (AT; 67.5 ng; magenta and green, respectively).

Techniques Used: Labeling, Purification, Mutagenesis

Related Articles

Recombinant:

Article Title: Identification of an Allosteric Binding Site on Human Lysosomal Alpha-Galactosidase Opens the Way to New Pharmacological Chaperones for Fabry Disease
Article Snippet: Melting profiles of recombinant human phosphomannomutase2 were recorded similarly, the only exception was the presence of 1mM MgCl2 in the buffer. .. In order to verify the reversibility of the effect of DTP on Fabrazyme® , the enzyme equilibrated in Na-Hepes 20 mM, NaCl 150 mM, pH 7.4 was incubated for 1 hour at 4°C in the presence of 6 mM DTP dissolved in DMSO, then the sample was dialysed by ultrafiltration (by using Centrifugal ultrafiltration unit 15-mL MWCO 10 kDa, Merck-Millipore).

Filtration:

Article Title: Solution Properties of Tetramethylrhodamine-Modified G-Actin
Article Snippet: DTT and HEPES were from Merck (Darmstadt, Germany). .. PD-10 gel filtration columns were purchased from Amersham Pharmacia Biotech (Uppsala, Sweden).

Positive Control:

Article Title: Antibacterial activity of chitosan against Burkholderia pseudomallei. Antibacterial activity of chitosan against Burkholderia pseudomallei
Article Snippet: .. Triton® X‐100 (Merck, KGaA, Darmstadt, Germany) at a concentration of 0.01% (v/v) was used as a positive control. .. 2.5 Transmission electron microscopy One milliliter of B. pseudomallei ST‐39 suspension of approximately 108 cfu ml−1 was added into sublethal chitosan solutions with final concentrations of 1 and 2 mg ml−1 .

Protease Inhibitor:

Article Title: Analysis of the substrate recognition state of TDP-43 to single-stranded DNA using fluorescence correlation spectroscopy
Article Snippet: Harvested cells were lysed in buffer A (50 mM HEPES-KOH (pH 7.5), 150 mM NaCl, and 1 mM dithiothreitol (DTT)) containing 0.1% Triton-X 100 and protease inhibitor cocktail (TaKaRa, Shiga, Japan), and ultracentrifuged at 40,000 rpm for 1 h at 4 °C. .. The buffer was replaced with 50 mM HEPES-KOH (pH 7.5) using a centrifugal dialysis filter (Amicon Ultra; Merck, Darmstadt, Germany), then 1% NP-40 was added if necessary.

Incubation:

Article Title: Identification of an Allosteric Binding Site on Human Lysosomal Alpha-Galactosidase Opens the Way to New Pharmacological Chaperones for Fabry Disease
Article Snippet: .. In order to verify the reversibility of the effect of DTP on Fabrazyme® , the enzyme equilibrated in Na-Hepes 20 mM, NaCl 150 mM, pH 7.4 was incubated for 1 hour at 4°C in the presence of 6 mM DTP dissolved in DMSO, then the sample was dialysed by ultrafiltration (by using Centrifugal ultrafiltration unit 15-mL MWCO 10 kDa, Merck-Millipore). ..

Cell Culture:

Article Title: Optimizing adipogenic transdifferentiation of bovine mesenchymal stem cells: a prominent role of ascorbic acid in FABP4 induction
Article Snippet: .. Materials FBS, BSL (Ex-Cyte), trypsin-EDTA, penicillin-streptomycin, AcA, Dulbecco’s phosphate-buffered saline (DPBS), collagen-A, and the cell culture media DMEM and complete α minimum essential medium (α-MEM) were obtained from Merck Millipore (Darmstadt, Germany). .. N-2-Hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES), amphotericin B, AsA, biotin, pantothenic acid, bovine insulin, rosiglitazone, dexamethasone, IBMX, Nile red, TritonTM X-100, gelatin-A, poly-L-lysine, ß-glycerophosphate, 1,25-dihydroxyvitamin D3 , alizarin red (sodium sulphonate salt) and D-glucose were purchased from Sigma Aldrich (Taufkirchen, Germany).

Article Title: Analysis of the substrate recognition state of TDP-43 to single-stranded DNA using fluorescence correlation spectroscopy
Article Snippet: When the optical density reached 0.45–0.5, the cells were cultured in medium containing 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 2 h at 18 °C to induce protein expression. .. The buffer was replaced with 50 mM HEPES-KOH (pH 7.5) using a centrifugal dialysis filter (Amicon Ultra; Merck, Darmstadt, Germany), then 1% NP-40 was added if necessary.

Purification:

Article Title: Identification of an Allosteric Binding Site on Human Lysosomal Alpha-Galactosidase Opens the Way to New Pharmacological Chaperones for Fabry Disease
Article Snippet: Phosphomannomutase 2 had been expressed and purified as described [ ]. .. In order to verify the reversibility of the effect of DTP on Fabrazyme® , the enzyme equilibrated in Na-Hepes 20 mM, NaCl 150 mM, pH 7.4 was incubated for 1 hour at 4°C in the presence of 6 mM DTP dissolved in DMSO, then the sample was dialysed by ultrafiltration (by using Centrifugal ultrafiltration unit 15-mL MWCO 10 kDa, Merck-Millipore).

Article Title: Analysis of the substrate recognition state of TDP-43 to single-stranded DNA using fluorescence correlation spectroscopy
Article Snippet: Paragraph title: 2.1. Protein expression and purification ... The buffer was replaced with 50 mM HEPES-KOH (pH 7.5) using a centrifugal dialysis filter (Amicon Ultra; Merck, Darmstadt, Germany), then 1% NP-40 was added if necessary.

Concentration Assay:

Article Title: Identification of an Allosteric Binding Site on Human Lysosomal Alpha-Galactosidase Opens the Way to New Pharmacological Chaperones for Fabry Disease
Article Snippet: The protein (0.5–1 mg/mL final concentration) was equilibrated in Na-Hepes 20 mM, NaCl 150 mM, pH 7.4 with Sypro Orange 2.5X (Invitrogen Molecular Probes, lifetechnologies.com), either without ligands or in the presence of ligands (6 mM DTP, 0.040 mM DGJ (SIGMA, Milan, Italy). .. In order to verify the reversibility of the effect of DTP on Fabrazyme® , the enzyme equilibrated in Na-Hepes 20 mM, NaCl 150 mM, pH 7.4 was incubated for 1 hour at 4°C in the presence of 6 mM DTP dissolved in DMSO, then the sample was dialysed by ultrafiltration (by using Centrifugal ultrafiltration unit 15-mL MWCO 10 kDa, Merck-Millipore).

Article Title: Antibacterial activity of chitosan against Burkholderia pseudomallei. Antibacterial activity of chitosan against Burkholderia pseudomallei
Article Snippet: .. Triton® X‐100 (Merck, KGaA, Darmstadt, Germany) at a concentration of 0.01% (v/v) was used as a positive control. .. 2.5 Transmission electron microscopy One milliliter of B. pseudomallei ST‐39 suspension of approximately 108 cfu ml−1 was added into sublethal chitosan solutions with final concentrations of 1 and 2 mg ml−1 .

Article Title: LIPID CONTENT OF BRAIN, OF BRAIN MEMBRANE LIPID DOMAINS, AND OF NEURONS FROM ACID SPHINGOMYELINASE DEFICIENT MICE (ASMKO)
Article Snippet: .. Different final protein/detergent ratios were obtained adding Triton X-100 in TNEV to 1 to 6 mg of protein homogenates, to reach a final concentration of 1% Triton X-100 in 1 mL. ..

Enzymatic Assay:

Article Title: Identification of an Allosteric Binding Site on Human Lysosomal Alpha-Galactosidase Opens the Way to New Pharmacological Chaperones for Fabry Disease
Article Snippet: In order to verify the reversibility of the effect of DTP on Fabrazyme® , the enzyme equilibrated in Na-Hepes 20 mM, NaCl 150 mM, pH 7.4 was incubated for 1 hour at 4°C in the presence of 6 mM DTP dissolved in DMSO, then the sample was dialysed by ultrafiltration (by using Centrifugal ultrafiltration unit 15-mL MWCO 10 kDa, Merck-Millipore). .. Each dialysed sample was analysed by enzymatic assay (at pH 7.5) and by TSA (the experiment was conducted either in the presence of 6 mM DTP or with only DMSO) as described above.

other:

Article Title: PIP2 signaling in lipid domains: a critical re-evaluation
Article Snippet: The measured lifetime of GFP alone was 2.4 ns, and the FRET efficiency E was calculated as FRET efficiencies from large populations (80–150 cells per experiment) were measured before and after treatment with CD (10 mM) or Triton X-100.

Article Title: Purification and Characterization of Lipase Produced by Leuconostoc mesenteroides Subsp. mesenteroides ATCC 8293 Using an Aqueous Two-Phase System (ATPS) Composed of Triton X-100 and Maltitol
Article Snippet: Gum Arabic, Triton X-100, isopropanol, Tween 80, and sodium chloride were bought from Merck (Darmstadt, Germany).

Article Title: Dye decolorization and detoxification potential of Ca-alginate beads immobilized manganese peroxidase
Article Snippet: Triton X-100 and cyclophosphamide were purchased from Merck (Germany) and Scharlau (Spain), respectively.

Article Title: LIPID CONTENT OF BRAIN, OF BRAIN MEMBRANE LIPID DOMAINS, AND OF NEURONS FROM ACID SPHINGOMYELINASE DEFICIENT MICE (ASMKO)
Article Snippet: Triton X-100 was from Merck (Darmstadt, Germany).

Expressing:

Article Title: Analysis of the substrate recognition state of TDP-43 to single-stranded DNA using fluorescence correlation spectroscopy
Article Snippet: Paragraph title: 2.1. Protein expression and purification ... The buffer was replaced with 50 mM HEPES-KOH (pH 7.5) using a centrifugal dialysis filter (Amicon Ultra; Merck, Darmstadt, Germany), then 1% NP-40 was added if necessary.

Polymerase Chain Reaction:

Article Title: Identification of an Allosteric Binding Site on Human Lysosomal Alpha-Galactosidase Opens the Way to New Pharmacological Chaperones for Fabry Disease
Article Snippet: The samples were distributed in 48-well PCR plates (0.025 mL in each well), sealed and heated from 20 to 90° at 1°C/min with increments of 0.6°C. .. In order to verify the reversibility of the effect of DTP on Fabrazyme® , the enzyme equilibrated in Na-Hepes 20 mM, NaCl 150 mM, pH 7.4 was incubated for 1 hour at 4°C in the presence of 6 mM DTP dissolved in DMSO, then the sample was dialysed by ultrafiltration (by using Centrifugal ultrafiltration unit 15-mL MWCO 10 kDa, Merck-Millipore).

Western Blot:

Article Title: Proton NMR visible mobile lipid signals in sensitive and multidrug-resistant K562 cells are modulated by rafts
Article Snippet: Chemicals Methyl-β-cyclodextrin, sphingomyelinase, cholesterol, chloroform, isopropanol and paraformaldehyde, as well as deuterium oxide (D2 O) and all chemicals for western-blot analysis were provided by Sigma-Aldrich (Saint-Quentin Fallavier, France). .. Triton X-100 was supplied by Merck (Schuchardt, Darmstadt, Germany), and methanol by Acros Organics (Geel, Belgium).

Binding Assay:

Article Title: Optimizing adipogenic transdifferentiation of bovine mesenchymal stem cells: a prominent role of ascorbic acid in FABP4 induction
Article Snippet: Materials FBS, BSL (Ex-Cyte), trypsin-EDTA, penicillin-streptomycin, AcA, Dulbecco’s phosphate-buffered saline (DPBS), collagen-A, and the cell culture media DMEM and complete α minimum essential medium (α-MEM) were obtained from Merck Millipore (Darmstadt, Germany). .. The antibodies against NT5E, THY1, delta like non-canonical notch ligand 1 (DLK1), fatty acid binding protein 4 (FABP4), glucose transporter type 4 (GLUT4) and fatty acid synthase (FAS) were from AbCam (Cambridge, UK), whereas the antibody against platelet derived growth factor receptor α (PDGFRα) was obtained from Biorbyt Ltd. (Cambridge, UK).

Derivative Assay:

Article Title: Optimizing adipogenic transdifferentiation of bovine mesenchymal stem cells: a prominent role of ascorbic acid in FABP4 induction
Article Snippet: Materials FBS, BSL (Ex-Cyte), trypsin-EDTA, penicillin-streptomycin, AcA, Dulbecco’s phosphate-buffered saline (DPBS), collagen-A, and the cell culture media DMEM and complete α minimum essential medium (α-MEM) were obtained from Merck Millipore (Darmstadt, Germany). .. The antibodies against NT5E, THY1, delta like non-canonical notch ligand 1 (DLK1), fatty acid binding protein 4 (FABP4), glucose transporter type 4 (GLUT4) and fatty acid synthase (FAS) were from AbCam (Cambridge, UK), whereas the antibody against platelet derived growth factor receptor α (PDGFRα) was obtained from Biorbyt Ltd. (Cambridge, UK).

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  • 92
    Merck KGaA na hepes
    2–6 dithiopurine stabilizes human lysosomal alpha-galactosidase in thermal shift assay. Panel A. Fabrazyme ® (in <t>Na-Hepes</t> 20 mM, NaCl 150 mM, pH 7.4) was equilibrated in the presence of ligands dissolved in DMSO 20%: DGJ 1 microM (empty triangle) or 40 microM (empty circle); <t>DTP</t> 6 mM (empty diamond); DTP 6 mM plus DGJ 1 microM (filled circle); DTP 6 mM plus DGJ 40 microM (filled triangle). A control (with only DMSO) was also shown (x). Panel B. Fabrazyme ® (in Na-Hepes 20 mM, NaCl 150 mM, pH 7.4) was incubated in the presence of DTP 6 mM for 1 hour at 4°C then DTP was eliminated by dialysis. A control experiment was conducted by incubating the enzyme only with DMSO. Both the aliquots of Fabrazyme ® were analysed by thermal shift assay in the presence of DTP 6 mM or in the presence of DMSO. Filled squares: DTP/DTP; open squares: DMSO/DTP; filled circles: DTP/DMSO; open circles: DMSO/DMSO where the first word of the label corresponds to the pretreatment and the second part corresponds to the presence of the compound during the thermal scan. The protein samples were heated from 20 to 90° at 1°C/min in the presence of Sypro Orange 2.5x. Data were shown as normalized curves.
    Na Hepes, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Merck KGaA hepes
    Nucleotide exchange inhibition by gelsolin-S1. Nucleotide exchange in the gelsolin-S1 complex with Ca-ATP-actin ( A ) and Mg-ADP-actin ( B ) was monitored via changes in fluorescence intensity upon ɛ -nucleotide release from, or incorporation into, the nucleotide-binding cleft on actin. 100 μ M ɛ -ATP ( A ) or ɛ -ADP ( B ) was added at the zero time to 2.0 μ M actin in 10 mM <t>HEPES</t> buffer, at pH 7.5, containing 1 mM <t>DTT,</t> and either 30 μ M CaCl 2 and 5.0 μ M ATP in A , or 30 μ M MgCl 2 and 5.0 μ M ADP in B . Etheno-nucleotide release was initiated by the addition of 1.0 mM ATP (marked by arrowheads above curves a and b ). Curve a , data in gray; no gelsolin-S1 present. Curve b , data in black; 4.0 μ M gelsolin-S1 was added to this sample after the fluorescence increase due to etheno-nucleotide incorporation reached a plateau (indicated by the arrow). Curve c , data in dark gray; 4.0 μ M gelsolin-S1 was added 5 min before the addition of ɛ -ATP or ɛ -ADP to G-actin (at the zero time). In all cases nucleotide exchange was abolished in the presence of gelsolin-S1.
    Hepes, supplied by Merck KGaA, 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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    94
    Merck KGaA mm hepes koh
    Autocorrelation functions (ACFs) of fluorescently labeled ssDNA in the presence and absence of purified TDP-43. Normalized ACFs of Alexa Fluor 647-labeled TG 12 (TG; A C) or T 20 (TT; B D) in 50 mM <t>HEPES-KOH</t> buffer without NP-40 are shown in the presence and absence of wild type TDP-43 (WT; 42.1 ng) or the A315T mutant (AT; 67.5 ng; magenta and green, respectively).
    Mm Hepes Koh, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Merck KGaA hepes naoh buffer
    pH-stability of FNLDH (a), PALDH (b), and ECLDH (c), and heat-stability of the enzymes (d). a-c) Each enzyme was treated at 30°C for 1 h in sodium citrate (open circles), sodium acetate (closed circles), <t>MES-NaOH</t> (open triangles), MOPS-NaOH (closed triangles), <t>HEPES-NaOH</t> (open squares), Bicine-NaOH ( N,N -Bis(2-hydroxyethyl)glycine) (closed squares), CHES-NaOH ( N -cyclohexyl-2-aminoethanesulfonic acid) (open diamonds), and CAPS-NaOH ( N -cyclohexyl-3-aminopropanesulfonic acid) (closed diamonds) buffers. d) FNLDH (white circles and solid lines), PALDH (grey circles and dashed lines), and ECLDH (black circles and dotted lines) were treated at various temperatures for 30 min in the buffers described in ‘Materials and methods’.
    Hepes Naoh Buffer, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    2–6 dithiopurine stabilizes human lysosomal alpha-galactosidase in thermal shift assay. Panel A. Fabrazyme ® (in Na-Hepes 20 mM, NaCl 150 mM, pH 7.4) was equilibrated in the presence of ligands dissolved in DMSO 20%: DGJ 1 microM (empty triangle) or 40 microM (empty circle); DTP 6 mM (empty diamond); DTP 6 mM plus DGJ 1 microM (filled circle); DTP 6 mM plus DGJ 40 microM (filled triangle). A control (with only DMSO) was also shown (x). Panel B. Fabrazyme ® (in Na-Hepes 20 mM, NaCl 150 mM, pH 7.4) was incubated in the presence of DTP 6 mM for 1 hour at 4°C then DTP was eliminated by dialysis. A control experiment was conducted by incubating the enzyme only with DMSO. Both the aliquots of Fabrazyme ® were analysed by thermal shift assay in the presence of DTP 6 mM or in the presence of DMSO. Filled squares: DTP/DTP; open squares: DMSO/DTP; filled circles: DTP/DMSO; open circles: DMSO/DMSO where the first word of the label corresponds to the pretreatment and the second part corresponds to the presence of the compound during the thermal scan. The protein samples were heated from 20 to 90° at 1°C/min in the presence of Sypro Orange 2.5x. Data were shown as normalized curves.

    Journal: PLoS ONE

    Article Title: Identification of an Allosteric Binding Site on Human Lysosomal Alpha-Galactosidase Opens the Way to New Pharmacological Chaperones for Fabry Disease

    doi: 10.1371/journal.pone.0165463

    Figure Lengend Snippet: 2–6 dithiopurine stabilizes human lysosomal alpha-galactosidase in thermal shift assay. Panel A. Fabrazyme ® (in Na-Hepes 20 mM, NaCl 150 mM, pH 7.4) was equilibrated in the presence of ligands dissolved in DMSO 20%: DGJ 1 microM (empty triangle) or 40 microM (empty circle); DTP 6 mM (empty diamond); DTP 6 mM plus DGJ 1 microM (filled circle); DTP 6 mM plus DGJ 40 microM (filled triangle). A control (with only DMSO) was also shown (x). Panel B. Fabrazyme ® (in Na-Hepes 20 mM, NaCl 150 mM, pH 7.4) was incubated in the presence of DTP 6 mM for 1 hour at 4°C then DTP was eliminated by dialysis. A control experiment was conducted by incubating the enzyme only with DMSO. Both the aliquots of Fabrazyme ® were analysed by thermal shift assay in the presence of DTP 6 mM or in the presence of DMSO. Filled squares: DTP/DTP; open squares: DMSO/DTP; filled circles: DTP/DMSO; open circles: DMSO/DMSO where the first word of the label corresponds to the pretreatment and the second part corresponds to the presence of the compound during the thermal scan. The protein samples were heated from 20 to 90° at 1°C/min in the presence of Sypro Orange 2.5x. Data were shown as normalized curves.

    Article Snippet: In order to verify the reversibility of the effect of DTP on Fabrazyme® , the enzyme equilibrated in Na-Hepes 20 mM, NaCl 150 mM, pH 7.4 was incubated for 1 hour at 4°C in the presence of 6 mM DTP dissolved in DMSO, then the sample was dialysed by ultrafiltration (by using Centrifugal ultrafiltration unit 15-mL MWCO 10 kDa, Merck-Millipore).

    Techniques: Thermal Shift Assay, Incubation

    Nucleotide exchange inhibition by gelsolin-S1. Nucleotide exchange in the gelsolin-S1 complex with Ca-ATP-actin ( A ) and Mg-ADP-actin ( B ) was monitored via changes in fluorescence intensity upon ɛ -nucleotide release from, or incorporation into, the nucleotide-binding cleft on actin. 100 μ M ɛ -ATP ( A ) or ɛ -ADP ( B ) was added at the zero time to 2.0 μ M actin in 10 mM HEPES buffer, at pH 7.5, containing 1 mM DTT, and either 30 μ M CaCl 2 and 5.0 μ M ATP in A , or 30 μ M MgCl 2 and 5.0 μ M ADP in B . Etheno-nucleotide release was initiated by the addition of 1.0 mM ATP (marked by arrowheads above curves a and b ). Curve a , data in gray; no gelsolin-S1 present. Curve b , data in black; 4.0 μ M gelsolin-S1 was added to this sample after the fluorescence increase due to etheno-nucleotide incorporation reached a plateau (indicated by the arrow). Curve c , data in dark gray; 4.0 μ M gelsolin-S1 was added 5 min before the addition of ɛ -ATP or ɛ -ADP to G-actin (at the zero time). In all cases nucleotide exchange was abolished in the presence of gelsolin-S1.

    Journal: Biophysical Journal

    Article Title: Solution Properties of Tetramethylrhodamine-Modified G-Actin

    doi:

    Figure Lengend Snippet: Nucleotide exchange inhibition by gelsolin-S1. Nucleotide exchange in the gelsolin-S1 complex with Ca-ATP-actin ( A ) and Mg-ADP-actin ( B ) was monitored via changes in fluorescence intensity upon ɛ -nucleotide release from, or incorporation into, the nucleotide-binding cleft on actin. 100 μ M ɛ -ATP ( A ) or ɛ -ADP ( B ) was added at the zero time to 2.0 μ M actin in 10 mM HEPES buffer, at pH 7.5, containing 1 mM DTT, and either 30 μ M CaCl 2 and 5.0 μ M ATP in A , or 30 μ M MgCl 2 and 5.0 μ M ADP in B . Etheno-nucleotide release was initiated by the addition of 1.0 mM ATP (marked by arrowheads above curves a and b ). Curve a , data in gray; no gelsolin-S1 present. Curve b , data in black; 4.0 μ M gelsolin-S1 was added to this sample after the fluorescence increase due to etheno-nucleotide incorporation reached a plateau (indicated by the arrow). Curve c , data in dark gray; 4.0 μ M gelsolin-S1 was added 5 min before the addition of ɛ -ATP or ɛ -ADP to G-actin (at the zero time). In all cases nucleotide exchange was abolished in the presence of gelsolin-S1.

    Article Snippet: DTT and HEPES were from Merck (Darmstadt, Germany).

    Techniques: Inhibition, Fluorescence, Binding Assay

    Autocorrelation functions (ACFs) of fluorescently labeled ssDNA in the presence and absence of purified TDP-43. Normalized ACFs of Alexa Fluor 647-labeled TG 12 (TG; A C) or T 20 (TT; B D) in 50 mM HEPES-KOH buffer without NP-40 are shown in the presence and absence of wild type TDP-43 (WT; 42.1 ng) or the A315T mutant (AT; 67.5 ng; magenta and green, respectively).

    Journal: Biochemistry and Biophysics Reports

    Article Title: Analysis of the substrate recognition state of TDP-43 to single-stranded DNA using fluorescence correlation spectroscopy

    doi: 10.1016/j.bbrep.2018.03.009

    Figure Lengend Snippet: Autocorrelation functions (ACFs) of fluorescently labeled ssDNA in the presence and absence of purified TDP-43. Normalized ACFs of Alexa Fluor 647-labeled TG 12 (TG; A C) or T 20 (TT; B D) in 50 mM HEPES-KOH buffer without NP-40 are shown in the presence and absence of wild type TDP-43 (WT; 42.1 ng) or the A315T mutant (AT; 67.5 ng; magenta and green, respectively).

    Article Snippet: The buffer was replaced with 50 mM HEPES-KOH (pH 7.5) using a centrifugal dialysis filter (Amicon Ultra; Merck, Darmstadt, Germany), then 1% NP-40 was added if necessary.

    Techniques: Labeling, Purification, Mutagenesis

    pH-stability of FNLDH (a), PALDH (b), and ECLDH (c), and heat-stability of the enzymes (d). a-c) Each enzyme was treated at 30°C for 1 h in sodium citrate (open circles), sodium acetate (closed circles), MES-NaOH (open triangles), MOPS-NaOH (closed triangles), HEPES-NaOH (open squares), Bicine-NaOH ( N,N -Bis(2-hydroxyethyl)glycine) (closed squares), CHES-NaOH ( N -cyclohexyl-2-aminoethanesulfonic acid) (open diamonds), and CAPS-NaOH ( N -cyclohexyl-3-aminopropanesulfonic acid) (closed diamonds) buffers. d) FNLDH (white circles and solid lines), PALDH (grey circles and dashed lines), and ECLDH (black circles and dotted lines) were treated at various temperatures for 30 min in the buffers described in ‘Materials and methods’.

    Journal: AMB Express

    Article Title: Diverse allosteric and catalytic functions of tetrameric d-lactate dehydrogenases from three Gram-negative bacteria

    doi: 10.1186/s13568-014-0076-1

    Figure Lengend Snippet: pH-stability of FNLDH (a), PALDH (b), and ECLDH (c), and heat-stability of the enzymes (d). a-c) Each enzyme was treated at 30°C for 1 h in sodium citrate (open circles), sodium acetate (closed circles), MES-NaOH (open triangles), MOPS-NaOH (closed triangles), HEPES-NaOH (open squares), Bicine-NaOH ( N,N -Bis(2-hydroxyethyl)glycine) (closed squares), CHES-NaOH ( N -cyclohexyl-2-aminoethanesulfonic acid) (open diamonds), and CAPS-NaOH ( N -cyclohexyl-3-aminopropanesulfonic acid) (closed diamonds) buffers. d) FNLDH (white circles and solid lines), PALDH (grey circles and dashed lines), and ECLDH (black circles and dotted lines) were treated at various temperatures for 30 min in the buffers described in ‘Materials and methods’.

    Article Snippet: The buffer for FNLDH and ECLDH was changed to 5 mM HEPES-NaOH buffer (pH 8.0), and the buffer for PALDH to 5 mM sodium acetate buffer (pH 5.0) using Amicon Ultra 30,000 molecular weight cut-off (Merck Millipore).

    Techniques:

    pH dependence of kinetic parameters on pyruvate reduction. White, grey, and black circles indicate FNLDH, PALDH, and ECLDH, respectively. The k cat (a) , S 0.5 (b) , and k cat / S 0.5 data (c) are plotted logarithmically, and the Hill coefficient (d) is plotted linearly. The buffers used for the assay were sodium acetate buffer (pH 4.5, 5.0, and 5.5; circles), MES-NaOH buffer (pH 5.5, 6.0, and 6.5; triangles), MOPS-NaOH buffer (pH 6.5, 7.0, and 7.5; squares), HEPES-NaOH buffer (pH 7.0, 7.5 and 8.0; diamonds), and Bicine-NaOH buffer (pH 8.0, 8.5, and 9.0; hexagons). We adjusted the pH of each buffer solution prior to the addition of substrate and cofactor, and confirmed that the pH value is not affected even in the presence of high concentrations of substrate.

    Journal: AMB Express

    Article Title: Diverse allosteric and catalytic functions of tetrameric d-lactate dehydrogenases from three Gram-negative bacteria

    doi: 10.1186/s13568-014-0076-1

    Figure Lengend Snippet: pH dependence of kinetic parameters on pyruvate reduction. White, grey, and black circles indicate FNLDH, PALDH, and ECLDH, respectively. The k cat (a) , S 0.5 (b) , and k cat / S 0.5 data (c) are plotted logarithmically, and the Hill coefficient (d) is plotted linearly. The buffers used for the assay were sodium acetate buffer (pH 4.5, 5.0, and 5.5; circles), MES-NaOH buffer (pH 5.5, 6.0, and 6.5; triangles), MOPS-NaOH buffer (pH 6.5, 7.0, and 7.5; squares), HEPES-NaOH buffer (pH 7.0, 7.5 and 8.0; diamonds), and Bicine-NaOH buffer (pH 8.0, 8.5, and 9.0; hexagons). We adjusted the pH of each buffer solution prior to the addition of substrate and cofactor, and confirmed that the pH value is not affected even in the presence of high concentrations of substrate.

    Article Snippet: The buffer for FNLDH and ECLDH was changed to 5 mM HEPES-NaOH buffer (pH 8.0), and the buffer for PALDH to 5 mM sodium acetate buffer (pH 5.0) using Amicon Ultra 30,000 molecular weight cut-off (Merck Millipore).

    Techniques: