β mercaptoethanol  (Millipore)


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    Millipore β mercaptoethanol
    Yield of the derivatization reaction using MSTFA/NH4I/ β -mercaptoethanol (a) and MSTFA (b) under different temperature and time periods.
    β Mercaptoethanol, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/β mercaptoethanol/product/Millipore
    Average 99 stars, based on 34 article reviews
    Price from $9.99 to $1999.99
    β mercaptoethanol - by Bioz Stars, 2020-05
    99/100 stars

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    Images

    1) Product Images from "Development of Sensitive and Specific Analysis of Vildagliptin in Pharmaceutical Formulation by Gas Chromatography-Mass Spectrometry"

    Article Title: Development of Sensitive and Specific Analysis of Vildagliptin in Pharmaceutical Formulation by Gas Chromatography-Mass Spectrometry

    Journal: Journal of Analytical Methods in Chemistry

    doi: 10.1155/2015/707414

    Yield of the derivatization reaction using MSTFA/NH4I/ β -mercaptoethanol (a) and MSTFA (b) under different temperature and time periods.
    Figure Legend Snippet: Yield of the derivatization reaction using MSTFA/NH4I/ β -mercaptoethanol (a) and MSTFA (b) under different temperature and time periods.

    Techniques Used:

    2) Product Images from "In vitro Conversion of Vinyl to Formyl Groups in Naturally Occurring Chlorophylls"

    Article Title: In vitro Conversion of Vinyl to Formyl Groups in Naturally Occurring Chlorophylls

    Journal: Scientific Reports

    doi: 10.1038/srep06069

    pH dependence of Chl d chemical synthesis from Chl a . Chromatograms (spectrum maximum plots) of detergent solubilised Chl a reacted with β-mercaptoethanol and heme in a 50 mM Tris buffer at the indicated pH. Unreacted Chl a , Chl d and 3 1 -sulfoxide of β-mercaptoethanol derivatives (a_I and a_II) are marked. Proportions of the major products and remaining Chl a and Chl a ’ (the C13 2 -Chl a epimer) are indicated in the pie charts for each pH tested.
    Figure Legend Snippet: pH dependence of Chl d chemical synthesis from Chl a . Chromatograms (spectrum maximum plots) of detergent solubilised Chl a reacted with β-mercaptoethanol and heme in a 50 mM Tris buffer at the indicated pH. Unreacted Chl a , Chl d and 3 1 -sulfoxide of β-mercaptoethanol derivatives (a_I and a_II) are marked. Proportions of the major products and remaining Chl a and Chl a ’ (the C13 2 -Chl a epimer) are indicated in the pie charts for each pH tested.

    Techniques Used:

    Substitutions at the C3 position of ring A of Chl a mediated by β-mercaptoethanol. The C3 vinyl position of Chl a is targeted by the thiol group of β-mercaptoethanol yielding a sulfoxide derivative of [3 1 -β-mercaptoethanol] Chl a (product a_II), which can react further with β-mercaptoethanol to yield an unkown product (a_I) with a mass of a_II plus 76 Da. An alternate, competing reaction also targets the C3 vinyl group of Chl a , oxidatively cleaving it to yield [3-formyl]-Chl a (Chl d ). As is the case for product a_II, [3-formyl]-Chl a also reacts further with β-mercaptoethanol to yield an unknown product (a_III) with a mass of [3-formyl]-Chl a plus 76 Da. Structure of product a_II and synthesized Chl d is confirmed by NMR anaylsis (details see supplementary information ).
    Figure Legend Snippet: Substitutions at the C3 position of ring A of Chl a mediated by β-mercaptoethanol. The C3 vinyl position of Chl a is targeted by the thiol group of β-mercaptoethanol yielding a sulfoxide derivative of [3 1 -β-mercaptoethanol] Chl a (product a_II), which can react further with β-mercaptoethanol to yield an unkown product (a_I) with a mass of a_II plus 76 Da. An alternate, competing reaction also targets the C3 vinyl group of Chl a , oxidatively cleaving it to yield [3-formyl]-Chl a (Chl d ). As is the case for product a_II, [3-formyl]-Chl a also reacts further with β-mercaptoethanol to yield an unknown product (a_III) with a mass of [3-formyl]-Chl a plus 76 Da. Structure of product a_II and synthesized Chl d is confirmed by NMR anaylsis (details see supplementary information ).

    Techniques Used: Synthesized, Nuclear Magnetic Resonance

    Reaction of monovinyl chlorophylls with β-mercaptoethanol in aqueous and methanolic environments. (a) Spectrum maximum (maximum absorbance reading at the range of 630–710 nm) RP-HPLC chromatograms of the aqueous buffer reaction mixture after reaction with β-mercaptoethanol using purified Chl a , Chl d , Chl b or Chl f as reactant. Dotted vertical lines mark the retention time of product a_III and Chl d . For more details regarding products a_I, a_II, a_III, b_I and f_I refer to the text and Table 1 . (b) RP-HPLC chromatograms of Chl a prior to (top panel) and following (two middle panels) reaction with β-mercaptoethanol in methanol, detected at indicated wavelengths. Chromatogram of Chl d from Acaryochloris (lower chromatogram) is shown for comparison with the chemically synthesised [3-formyl]-Chl a (Chl d ). Dotted vertical line marks the retention time of Chl d and Chl a . (c) Online spectra of the major derivatives synthesised from Chl a . The absorption spectra of Chl a and Chl d are plotted and their Q y maxima are marked with vertical lines for comparison with synthesised products. Q y maxima are indicated (in nm).
    Figure Legend Snippet: Reaction of monovinyl chlorophylls with β-mercaptoethanol in aqueous and methanolic environments. (a) Spectrum maximum (maximum absorbance reading at the range of 630–710 nm) RP-HPLC chromatograms of the aqueous buffer reaction mixture after reaction with β-mercaptoethanol using purified Chl a , Chl d , Chl b or Chl f as reactant. Dotted vertical lines mark the retention time of product a_III and Chl d . For more details regarding products a_I, a_II, a_III, b_I and f_I refer to the text and Table 1 . (b) RP-HPLC chromatograms of Chl a prior to (top panel) and following (two middle panels) reaction with β-mercaptoethanol in methanol, detected at indicated wavelengths. Chromatogram of Chl d from Acaryochloris (lower chromatogram) is shown for comparison with the chemically synthesised [3-formyl]-Chl a (Chl d ). Dotted vertical line marks the retention time of Chl d and Chl a . (c) Online spectra of the major derivatives synthesised from Chl a . The absorption spectra of Chl a and Chl d are plotted and their Q y maxima are marked with vertical lines for comparison with synthesised products. Q y maxima are indicated (in nm).

    Techniques Used: High Performance Liquid Chromatography, Purification

    Dithiothreitol and benzyl mercaptan also facilitate the conversion of the C3 vinyl of Chl a (a) and Chl b (b) to a formyl. RP-HPLC chromatograms (top panels) demonstrate retention times of the 3 1 -formyl derivatives of Chl a and Chl b were identical and are marked with vertical lines. Spectra (bottom panels) were identical to the 3 1 -formyl derivatives formed in the presence of β-mercaptoethanol (β-merc.). Note, in the case of the reaction of Chl a with benzyl mercaptan, another product spectrally similar to Chl a has a similar retention time to Chl d , causing the mixed online absorption spectrum. Q y for [3-formyl]-Chl a (Chl d ) and [3-formyl]-Chl b are marked. Dotted line, β-mercaptoethanol; dashed line, dithiothreitol; solid line, benzyl mercaptan.
    Figure Legend Snippet: Dithiothreitol and benzyl mercaptan also facilitate the conversion of the C3 vinyl of Chl a (a) and Chl b (b) to a formyl. RP-HPLC chromatograms (top panels) demonstrate retention times of the 3 1 -formyl derivatives of Chl a and Chl b were identical and are marked with vertical lines. Spectra (bottom panels) were identical to the 3 1 -formyl derivatives formed in the presence of β-mercaptoethanol (β-merc.). Note, in the case of the reaction of Chl a with benzyl mercaptan, another product spectrally similar to Chl a has a similar retention time to Chl d , causing the mixed online absorption spectrum. Q y for [3-formyl]-Chl a (Chl d ) and [3-formyl]-Chl b are marked. Dotted line, β-mercaptoethanol; dashed line, dithiothreitol; solid line, benzyl mercaptan.

    Techniques Used: High Performance Liquid Chromatography

    RP-HPLC chromatogram and online absorption spectra of 8-vinyl Chl a formyl and sulfoxide of β-mercaptoethanol derivatives. (a) RP-HPLC spectrum maximum plot of an incomplete 8-vinyl Chl a reaction after 2 h. Products with either a C3 or C8 conversion (one conversion) are marked in blue, and those products where both the C3 and C8 vinyl have reacted (two conversions) are marked with red. (b) Online spectra of 8-vinyl Chl a and the major formyl and sulfoxide of β-mercaptoethanol derivatives. The Soret and Q y maxima of 8-vinyl Chl a are marked with vertical red lines. Spectra are arithmetically shifted for clarity. βME, sulfoxide of β-mercaptoethanol.
    Figure Legend Snippet: RP-HPLC chromatogram and online absorption spectra of 8-vinyl Chl a formyl and sulfoxide of β-mercaptoethanol derivatives. (a) RP-HPLC spectrum maximum plot of an incomplete 8-vinyl Chl a reaction after 2 h. Products with either a C3 or C8 conversion (one conversion) are marked in blue, and those products where both the C3 and C8 vinyl have reacted (two conversions) are marked with red. (b) Online spectra of 8-vinyl Chl a and the major formyl and sulfoxide of β-mercaptoethanol derivatives. The Soret and Q y maxima of 8-vinyl Chl a are marked with vertical red lines. Spectra are arithmetically shifted for clarity. βME, sulfoxide of β-mercaptoethanol.

    Techniques Used: High Performance Liquid Chromatography

    3) Product Images from "Vibrational Detection of Odorant Functional Groups by Drosophila melanogaster"

    Article Title: Vibrational Detection of Odorant Functional Groups by Drosophila melanogaster

    Journal: eNeuro

    doi: 10.1523/ENEURO.0049-17.2017

    Responses to β-mercaptoethanol and decaborane in wild-type Orco − and Ir8a − Orco − double mutants. A , The high molecular divergence of β-mercaptoethanol and decaborane. B–D , All graphs represent the mean relative distribution of flies in the arms of the maze ± SEM after the indicated treatment. Groups of flies were exposed to the indicated odorants per condition. Significance values are calculated by LSM contrast analysis and are indicated on the graphs with a single star. B , Responses of wild-type and anosmic flies to a range of β-mercaptoethanol concentrations. Wild-type flies are shown in white bars, Orco − flies are shown in light gray bars and Ir8a;Orco - flies are shown in dark gray bars. ANOVA indicated significant differences ( F (6,87) = 39.7679, p
    Figure Legend Snippet: Responses to β-mercaptoethanol and decaborane in wild-type Orco − and Ir8a − Orco − double mutants. A , The high molecular divergence of β-mercaptoethanol and decaborane. B–D , All graphs represent the mean relative distribution of flies in the arms of the maze ± SEM after the indicated treatment. Groups of flies were exposed to the indicated odorants per condition. Significance values are calculated by LSM contrast analysis and are indicated on the graphs with a single star. B , Responses of wild-type and anosmic flies to a range of β-mercaptoethanol concentrations. Wild-type flies are shown in white bars, Orco − flies are shown in light gray bars and Ir8a;Orco - flies are shown in dark gray bars. ANOVA indicated significant differences ( F (6,87) = 39.7679, p

    Techniques Used:

    After conditioning Drosophila do not discriminate decaborane from β-mercaptoethanol. All graphs represent the mean relative distribution of flies in the arms of the maze ± SEM after the indicated treatment. Significant differences are calculated relative to the performance of naïve animals (open bars) by LSM contrast analysis and are indicated on the graphs with a single star. A , ANOVA indicated significant differences ( F (2,59) = 8.5292, p = 0.0006). Flies shocked in the presence of β-mercaptoethanol augmented their avoidance toward it (light gray bar, p = 0.0005) and surprisingly a similar augmentation was shown by animals shocked in the presence of decaborane (dark bar, p = 0.0006). Indeed, the performances of the two differently trained groups were not significantly different ( p = 0.5915). n ≥ 12 for all conditions, with a total of 900-2000 flies per condition. B , In the converse experiment ANOVA indicated significant differences ( F (2,30) = 15.3343, p
    Figure Legend Snippet: After conditioning Drosophila do not discriminate decaborane from β-mercaptoethanol. All graphs represent the mean relative distribution of flies in the arms of the maze ± SEM after the indicated treatment. Significant differences are calculated relative to the performance of naïve animals (open bars) by LSM contrast analysis and are indicated on the graphs with a single star. A , ANOVA indicated significant differences ( F (2,59) = 8.5292, p = 0.0006). Flies shocked in the presence of β-mercaptoethanol augmented their avoidance toward it (light gray bar, p = 0.0005) and surprisingly a similar augmentation was shown by animals shocked in the presence of decaborane (dark bar, p = 0.0006). Indeed, the performances of the two differently trained groups were not significantly different ( p = 0.5915). n ≥ 12 for all conditions, with a total of 900-2000 flies per condition. B , In the converse experiment ANOVA indicated significant differences ( F (2,30) = 15.3343, p

    Techniques Used:

    4) Product Images from "Self-Association Process of a Peptide in Solution: From β-Sheet Filaments to Large Embedded Nanotubes"

    Article Title: Self-Association Process of a Peptide in Solution: From β-Sheet Filaments to Large Embedded Nanotubes

    Journal: Biophysical Journal

    doi:

    FTIR spectra in the frequencies of amide I vibrations ( a and b ) and FT-Raman spectra ( c – e ) measured at 20°C for 5% ( 1 ), 10% ( 2 ), and 15% ( 3 ) (w/w) Lanreotide acetate in water. The spectra 1′ and 2′ were obtained for Lanreotide concentration similar to the spectra 1 and 2, respectively, but in presence of β -mercaptoethanol (14 M). The spectrum ( 4 ) on d has been recorded for pure β -mercaptoethanol. The spectra 5 and 6 on e have been recorded for pure naphthalene and naphthalene in tetrahydrofurane, respectively.
    Figure Legend Snippet: FTIR spectra in the frequencies of amide I vibrations ( a and b ) and FT-Raman spectra ( c – e ) measured at 20°C for 5% ( 1 ), 10% ( 2 ), and 15% ( 3 ) (w/w) Lanreotide acetate in water. The spectra 1′ and 2′ were obtained for Lanreotide concentration similar to the spectra 1 and 2, respectively, but in presence of β -mercaptoethanol (14 M). The spectrum ( 4 ) on d has been recorded for pure β -mercaptoethanol. The spectra 5 and 6 on e have been recorded for pure naphthalene and naphthalene in tetrahydrofurane, respectively.

    Techniques Used: Concentration Assay

    5) Product Images from "The Escherichia coli CydX Protein Is a Member of the CydAB Cytochrome bd Oxidase Complex and Is Required for Cytochrome bd Oxidase Activity"

    Article Title: The Escherichia coli CydX Protein Is a Member of the CydAB Cytochrome bd Oxidase Complex and Is Required for Cytochrome bd Oxidase Activity

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00324-13

    Phenotypes of the Δ cydX mutant. (A) Growth curve of E. coli MG1655 and Δ cydX strains grown under aerobic conditions. Wild-type and mutant strains were grown with arabinose (+Ara) or without arabinose (-Ara) in order to control for nonspecific effects of arabinose on growth. E. coli MG1655 grown with and without arabinose, the Δ cydX mutant grown with and without arabinose, the Δ cydX mutant transformed with the pBAD24 plasmid grown without arabinose, and the Δ cydX mutant transformed with the pBAD24- cydX plasmid grown with arabinose are shown. Cultures were inoculated from overnight cultures with an equal number of cells as determined spectroscopically and grown at 37°C. (B) Zones of inhibition caused by the reductant β-mercaptoethanol. Overnight cultures of each strain were diluted (normalized to the OD 600 ) in top agar and plated on LB plates. A filter disk was placed in the center of each plate, and 10 μl of concentrated β-mercaptoethanol was added to each disk. Plates were then incubated at 30°C overnight. Values are average sizes (in centimeters) of the zone of inhibition plus standard error (error bars) from 4 to 12 plates. (C) Streaks to single colonies of E. coli MG1655 and Δ cydABXE and Δ cydX mutant cells showing mixed-colony formation in the mutant strains. Streaks are from single colonies grown on LB plates at 37°C overnight.
    Figure Legend Snippet: Phenotypes of the Δ cydX mutant. (A) Growth curve of E. coli MG1655 and Δ cydX strains grown under aerobic conditions. Wild-type and mutant strains were grown with arabinose (+Ara) or without arabinose (-Ara) in order to control for nonspecific effects of arabinose on growth. E. coli MG1655 grown with and without arabinose, the Δ cydX mutant grown with and without arabinose, the Δ cydX mutant transformed with the pBAD24 plasmid grown without arabinose, and the Δ cydX mutant transformed with the pBAD24- cydX plasmid grown with arabinose are shown. Cultures were inoculated from overnight cultures with an equal number of cells as determined spectroscopically and grown at 37°C. (B) Zones of inhibition caused by the reductant β-mercaptoethanol. Overnight cultures of each strain were diluted (normalized to the OD 600 ) in top agar and plated on LB plates. A filter disk was placed in the center of each plate, and 10 μl of concentrated β-mercaptoethanol was added to each disk. Plates were then incubated at 30°C overnight. Values are average sizes (in centimeters) of the zone of inhibition plus standard error (error bars) from 4 to 12 plates. (C) Streaks to single colonies of E. coli MG1655 and Δ cydABXE and Δ cydX mutant cells showing mixed-colony formation in the mutant strains. Streaks are from single colonies grown on LB plates at 37°C overnight.

    Techniques Used: Mutagenesis, Acetylene Reduction Assay, Transformation Assay, Plasmid Preparation, Inhibition, Incubation

    Site-directed mutagenesis of CydX. Single-amino-acid mutations were created in the cydX gene expressed from the pBAD24 plasmid, and the mutant proteins were tested for their ability to complement the β-mercaptoethanol sensitivity phenotype of the Δ cydX mutant. Sensitivity to β-mercaptoethanol was measured using zone of inhibition assays in which overnight cultures were diluted into top agar and spread onto plates containing 100 μg/ml ampicillin and 0.2% arabinose. After the plates had cooled, a filter disk was set in the middle of the plate, and 10 μl of β-mercaptoethanol was added to the disk. The plates were incubated at 30°C overnight, and zones of growth inhibition were measured the next day. Values represent average zone size plus standard error (in centimeters) from 4 to 8 plates.
    Figure Legend Snippet: Site-directed mutagenesis of CydX. Single-amino-acid mutations were created in the cydX gene expressed from the pBAD24 plasmid, and the mutant proteins were tested for their ability to complement the β-mercaptoethanol sensitivity phenotype of the Δ cydX mutant. Sensitivity to β-mercaptoethanol was measured using zone of inhibition assays in which overnight cultures were diluted into top agar and spread onto plates containing 100 μg/ml ampicillin and 0.2% arabinose. After the plates had cooled, a filter disk was set in the middle of the plate, and 10 μl of β-mercaptoethanol was added to the disk. The plates were incubated at 30°C overnight, and zones of growth inhibition were measured the next day. Values represent average zone size plus standard error (in centimeters) from 4 to 8 plates.

    Techniques Used: Mutagenesis, Plasmid Preparation, Inhibition, Incubation

    6) Product Images from "Autophagy activator promotes neuronal differentiation of adult adipose-derived stromal cells ★"

    Article Title: Autophagy activator promotes neuronal differentiation of adult adipose-derived stromal cells ★

    Journal: Neural Regeneration Research

    doi: 10.3969/j.issn.1673-5374.2013.10.002

    Identification of morphological changes in differentiated adult adipose-derived stromal cells in control and rapamycin groups after 5 mM β-mercaptoethanol induction, using inverted phase contrast microscopy (× 100). One hour after induction, axon-like processes were visible (arrows). Three hours after induction, cells showed neuronal-like changes (arrows). At 5 and 8 hours after induction, cells exhibited typical neuronal morphologies. Arrows indicate axon- and dendrite-like structures. In the rapamycin group, cell body enhanced refraction was observed.
    Figure Legend Snippet: Identification of morphological changes in differentiated adult adipose-derived stromal cells in control and rapamycin groups after 5 mM β-mercaptoethanol induction, using inverted phase contrast microscopy (× 100). One hour after induction, axon-like processes were visible (arrows). Three hours after induction, cells showed neuronal-like changes (arrows). At 5 and 8 hours after induction, cells exhibited typical neuronal morphologies. Arrows indicate axon- and dendrite-like structures. In the rapamycin group, cell body enhanced refraction was observed.

    Techniques Used: Derivative Assay, Microscopy

    Transmission electron microscope observation of the ultrastructure of normal adipose-derived stromal cell-differentiated neuronal-like cells at 5 hours after 5 mM β-mercaptoethanol induction. (A) A typical neuron. The arrow indicates a large round nucleus with a large amount of euchromatin but little heterochromatin (× 5 000). (B) Cytoplasmic ultrastructure. The arrow points to a large number of Nissl bodies (× 10 000). (C) Autophagy. The arrow indicates an autophagic cell with engulfed mitochondria. The arrowhead indicates a swollen mitochondria (× 10 000). (D) Arrow indicate autolysosomes (× 10 000).
    Figure Legend Snippet: Transmission electron microscope observation of the ultrastructure of normal adipose-derived stromal cell-differentiated neuronal-like cells at 5 hours after 5 mM β-mercaptoethanol induction. (A) A typical neuron. The arrow indicates a large round nucleus with a large amount of euchromatin but little heterochromatin (× 5 000). (B) Cytoplasmic ultrastructure. The arrow points to a large number of Nissl bodies (× 10 000). (C) Autophagy. The arrow indicates an autophagic cell with engulfed mitochondria. The arrowhead indicates a swollen mitochondria (× 10 000). (D) Arrow indicate autolysosomes (× 10 000).

    Techniques Used: Transmission Assay, Microscopy, Derivative Assay

    LC3 expression in 5 mM β-mercaptoethanol induced adult adipose-derived stromal cells in control and rapamycin groups (immunocytochemistry staining, light microscope, × 100). In the rapamycin group, LC3-positive expression is mainly concentrated around the nucleus, and more increasingly focused around the nucleus with extension of induction time. Arrows indicate LC3 positive cells with brown cytoplasm and a blue nucleus.
    Figure Legend Snippet: LC3 expression in 5 mM β-mercaptoethanol induced adult adipose-derived stromal cells in control and rapamycin groups (immunocytochemistry staining, light microscope, × 100). In the rapamycin group, LC3-positive expression is mainly concentrated around the nucleus, and more increasingly focused around the nucleus with extension of induction time. Arrows indicate LC3 positive cells with brown cytoplasm and a blue nucleus.

    Techniques Used: Expressing, Derivative Assay, Immunocytochemistry, Staining, Light Microscopy

    Expression of neuron specific enolase (NSE), neurofilament-200 (NF-200) and glial fibrillary acidic protein (GFAP) in adult adipose-derived stromal cells after 5 mM β-mercaptoethanol induction (immunocytochemistry staining, light microscope, × 100). Arrows indicate NF-200 and NSE positive cells. Expression of NF-200 and NSE increased gradually in the cytoplasm as the induction time extended and peaked at 5 hours. There was no GFAP expression after induction.
    Figure Legend Snippet: Expression of neuron specific enolase (NSE), neurofilament-200 (NF-200) and glial fibrillary acidic protein (GFAP) in adult adipose-derived stromal cells after 5 mM β-mercaptoethanol induction (immunocytochemistry staining, light microscope, × 100). Arrows indicate NF-200 and NSE positive cells. Expression of NF-200 and NSE increased gradually in the cytoplasm as the induction time extended and peaked at 5 hours. There was no GFAP expression after induction.

    Techniques Used: Expressing, Derivative Assay, Immunocytochemistry, Staining, Light Microscopy

    7) Product Images from "Self-assembly of keratin peptides: Its implication on the performance of electrospun PVA nanofibers"

    Article Title: Self-assembly of keratin peptides: Its implication on the performance of electrospun PVA nanofibers

    Journal: Scientific Reports

    doi: 10.1038/srep36558

    Bar plot of cell viability values of ( A ) Control ( B ) Sulphitolysis ( C ) β-Mercaptoethanol ( D ) Thioglycolic acid based Keratin/PVA nanofibrous mats at different time interval.
    Figure Legend Snippet: Bar plot of cell viability values of ( A ) Control ( B ) Sulphitolysis ( C ) β-Mercaptoethanol ( D ) Thioglycolic acid based Keratin/PVA nanofibrous mats at different time interval.

    Techniques Used:

    Temperature dependent CD spectra of keratin hydrolysates obtained by ( a ) Sulphitolysis ( b ) β-Mercaptoethanol ( c ) Thioglycolic acid based methods.
    Figure Legend Snippet: Temperature dependent CD spectra of keratin hydrolysates obtained by ( a ) Sulphitolysis ( b ) β-Mercaptoethanol ( c ) Thioglycolic acid based methods.

    Techniques Used:

    DSC thermograms of ( A ) Control ( B ) Sulphitolysis ( C ) β-Mercaptoethanol ( D ) Thioglycolic acid based keratin/PVA nanofibrous mats.
    Figure Legend Snippet: DSC thermograms of ( A ) Control ( B ) Sulphitolysis ( C ) β-Mercaptoethanol ( D ) Thioglycolic acid based keratin/PVA nanofibrous mats.

    Techniques Used:

    Bar plot of tensile strength values of ( A ) Control ( B ) Sulphitolysis ( C ) β-Mercaptoethanol ( D ) Thioglycolic acid based Keratin/PVA nanofibrous mats.
    Figure Legend Snippet: Bar plot of tensile strength values of ( A ) Control ( B ) Sulphitolysis ( C ) β-Mercaptoethanol ( D ) Thioglycolic acid based Keratin/PVA nanofibrous mats.

    Techniques Used:

    8) Product Images from "Apoptosis Signal-Regulating Kinase 1-Thioredoxin Complex Dissociation by Capsaicin Causes Pancreatic Tumor Growth Suppression by Inducing Apoptosis"

    Article Title: Apoptosis Signal-Regulating Kinase 1-Thioredoxin Complex Dissociation by Capsaicin Causes Pancreatic Tumor Growth Suppression by Inducing Apoptosis

    Journal: Antioxidants & Redox Signaling

    doi: 10.1089/ars.2011.4369

    Activation of ASK1 kinase activity by capsaicin is attenuable by a reducing agent and augmented by a Trx inhibitor. AsPC-1 cells were treated with (A) 50 μ M β-mercaptoethanol or (C) 25 μ M 1chloro 2,4-dinitro benzene
    Figure Legend Snippet: Activation of ASK1 kinase activity by capsaicin is attenuable by a reducing agent and augmented by a Trx inhibitor. AsPC-1 cells were treated with (A) 50 μ M β-mercaptoethanol or (C) 25 μ M 1chloro 2,4-dinitro benzene

    Techniques Used: Activation Assay, Activity Assay

    9) Product Images from "High-level production and purification in a functional state of an extrasynaptic gamma-aminobutyric acid type A receptor containing α4β3δ subunits"

    Article Title: High-level production and purification in a functional state of an extrasynaptic gamma-aminobutyric acid type A receptor containing α4β3δ subunits

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0191583

    Stability of the α4-subunit. ( A ) Western blot depicting fragmentation of α4-subunit seen as two bands in N-Flag-α4β3 and α4β3N-Flag-δ receptors reconstituted into CHAPS/asolectin micelles is presented. Both α4 bands are identified by polyclonal anti-α4 and monoclonal anti-Flag antibodies in the former receptor, confirming identity of the band. Numbers on the side indicate the position of the molecular weight markers (kDa). ( B ) Cells induced to express indicated GABA A Rs were prepared for Western blotting by either 1. suspending cells in suspension buffer; 2. suspending and sonicating; 3. leaving in a monolayer (untreated); or 4. lysing directly in the well with suspension buffer supplemented with 10 mM DDM; as indicated, prior to lysing cells with a 4x Laemmli sample buffer with 10% β-mercaptoethanol. Suspension buffer was supplemented with Protease Inhibitor Cocktail (Sigma) at 1:100 dilution. ( C ) Representative Western blot of samples obtained during α4β3N-Flag-δ receptor purification, as described in the materials and methods section. Numbers under each lane indicate the fraction the lower band comprises of the higher band, expressed as percentile points. ( D ) Membrane fraction from the α4β3N-Flag-δ was incubated for 1 hour at indicated temperatures prior to analysis by Western blotting. All blots are presented as grayscale and were uniformly adjusted for brightness and contrast to facilitate analysis. Full immunoblots used to make panels A-D are presented as S4 – S7 Figs.
    Figure Legend Snippet: Stability of the α4-subunit. ( A ) Western blot depicting fragmentation of α4-subunit seen as two bands in N-Flag-α4β3 and α4β3N-Flag-δ receptors reconstituted into CHAPS/asolectin micelles is presented. Both α4 bands are identified by polyclonal anti-α4 and monoclonal anti-Flag antibodies in the former receptor, confirming identity of the band. Numbers on the side indicate the position of the molecular weight markers (kDa). ( B ) Cells induced to express indicated GABA A Rs were prepared for Western blotting by either 1. suspending cells in suspension buffer; 2. suspending and sonicating; 3. leaving in a monolayer (untreated); or 4. lysing directly in the well with suspension buffer supplemented with 10 mM DDM; as indicated, prior to lysing cells with a 4x Laemmli sample buffer with 10% β-mercaptoethanol. Suspension buffer was supplemented with Protease Inhibitor Cocktail (Sigma) at 1:100 dilution. ( C ) Representative Western blot of samples obtained during α4β3N-Flag-δ receptor purification, as described in the materials and methods section. Numbers under each lane indicate the fraction the lower band comprises of the higher band, expressed as percentile points. ( D ) Membrane fraction from the α4β3N-Flag-δ was incubated for 1 hour at indicated temperatures prior to analysis by Western blotting. All blots are presented as grayscale and were uniformly adjusted for brightness and contrast to facilitate analysis. Full immunoblots used to make panels A-D are presented as S4 – S7 Figs.

    Techniques Used: Western Blot, Molecular Weight, Protease Inhibitor, Purification, Incubation

    10) Product Images from "Ubiquitin-like modifier FAT10 attenuates RIG-I mediated antiviral signaling by segregating activated RIG-I from its signaling platform"

    Article Title: Ubiquitin-like modifier FAT10 attenuates RIG-I mediated antiviral signaling by segregating activated RIG-I from its signaling platform

    Journal: Scientific Reports

    doi: 10.1038/srep23377

    Overexpressed FAT10 induces activated RIG-I (2CARD) precipitation. ( a ), HEK293FT cells were transfected with indicated plasmids for 48 h. Cells were treated with 20 μM MG132. ( b ), HEK293FT cells were transfected with indicated plasmids for 48 h. After harvesting, the soluble and insoluble fractions were prepared using RIPA lysis buffer. The protein levels of FAT10 and CARD in each fraction were analyzed. ( c ), HEK293FT cells were transfected with indicated plasmids for 48 h, whole cell lysates were prepared in Laemmli buffer containing 5% β-mercaptoethanol. ( d ), HEK293FT cells were transfected with indicated plasmids for 48 h, Lasota NDV was infected for 12 h before harvest. Cytosol (soluble) and insoluble fractions were prepared using sucrose buffer. Protein levels of FAT10 and RIG-I in each fraction were analyzed. ( e ), HepG2 cells were pre-treated with TNFα and IFNγ with final concentration (ng/ml) as indicated for 2 h following by Poly I:C (10 μg/ml) transfection. MG132 (20 μM) or DMSO was treated for 4 h before harvesting. Endogenous RIG-I and FAT10 protein levels were analyzed in the RIPA soluble and insoluble fractions. ( f ), same as e, except HepG2 cells were transfected with control or FAT10 siRNA. ( g ), HEK293FT cells were transfected with indicated plasmids, and the protein levels of FAT10 and CARD were examined in the mitochondria enriched fraction.
    Figure Legend Snippet: Overexpressed FAT10 induces activated RIG-I (2CARD) precipitation. ( a ), HEK293FT cells were transfected with indicated plasmids for 48 h. Cells were treated with 20 μM MG132. ( b ), HEK293FT cells were transfected with indicated plasmids for 48 h. After harvesting, the soluble and insoluble fractions were prepared using RIPA lysis buffer. The protein levels of FAT10 and CARD in each fraction were analyzed. ( c ), HEK293FT cells were transfected with indicated plasmids for 48 h, whole cell lysates were prepared in Laemmli buffer containing 5% β-mercaptoethanol. ( d ), HEK293FT cells were transfected with indicated plasmids for 48 h, Lasota NDV was infected for 12 h before harvest. Cytosol (soluble) and insoluble fractions were prepared using sucrose buffer. Protein levels of FAT10 and RIG-I in each fraction were analyzed. ( e ), HepG2 cells were pre-treated with TNFα and IFNγ with final concentration (ng/ml) as indicated for 2 h following by Poly I:C (10 μg/ml) transfection. MG132 (20 μM) or DMSO was treated for 4 h before harvesting. Endogenous RIG-I and FAT10 protein levels were analyzed in the RIPA soluble and insoluble fractions. ( f ), same as e, except HepG2 cells were transfected with control or FAT10 siRNA. ( g ), HEK293FT cells were transfected with indicated plasmids, and the protein levels of FAT10 and CARD were examined in the mitochondria enriched fraction.

    Techniques Used: Transfection, Lysis, Infection, Concentration Assay

    11) Product Images from "p35/Cyclin-Dependent Kinase 5 Phosphorylation of Ras Guanine Nucleotide Releasing Factor 2 (RasGRF2) Mediates Rac-Dependent Extracellular Signal-Regulated Kinase 1/2 Activity, Altering RasGRF2 and Microtubule-Associated Protein 1b Distribution in Neurons"

    Article Title: p35/Cyclin-Dependent Kinase 5 Phosphorylation of Ras Guanine Nucleotide Releasing Factor 2 (RasGRF2) Mediates Rac-Dependent Extracellular Signal-Regulated Kinase 1/2 Activity, Altering RasGRF2 and Microtubule-Associated Protein 1b Distribution in Neurons

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.0690-04.2004

    p35/Cdk5 and RasGRF2 mediate cellular Rac activity and ERK1/2 activity. A , CHO cells were transfected with EV-GTP (lane 1), RasGRF2 (lane 2), RasGRF2+p35+Cdk5 (lane 3), RasGRF2+Cdk5 (lane4), and EV+GTP (lane5). CHO cell lysates were incubated with GST-PAK1 and GST beads for 1 hr. Beads with bound active Rac were washed three times in lysis–binding–wash buffer and eluted by addition of 2× SDS-sample buffer/β-mercaptoethanol and boiling for 5 min. Eluted, active Rac samples were resolved by SDS-PAGE, transferred onto nitrocellulose, and immunodetected using the monoclonal anti-Rac1 Ab provided in the kit at 1:1000 dilution. p35/Cdk5 reduced RasGRF2-mediated active Rac levels. EV lysate incubated with GTP before the pull-downs exhibited the highest active Rac levels (EV+GTP). Representative Western blots show active Rac1 levels and their corresponding total Rac levels. B , p35/Cdk5 does not affect RasGRF2-mediated Ras activity. Transfections are as follows: 1, EV; 2, RasGRF2; 3, RasGRF2+p35+Cdk5; 4, RasGRF2+Cdk5; 5, EV+GTP. CHO cell lysates were incubated with GST-Raf1 and GST beads for 1 hr. Beads with bound active Ras were washed three times before addition of 2× SDS-sample buffer/β-mercaptoethanol and boiling for 5 min. Eluted active Ras samples were resolved by SDS-PAGE and transferred onto nitrocellulose, and Ras was detected with the monoclonal anti-ras Ab provided in the kit. Immunodetected samples are indicated next to the panels. C , CHO cells were cotransfected with the following: 1, EV; 2, RasGRF2; 3, RasGRF2+p35+Cdk5; 4, RasGRF2+Cdk5. Lysates were harvested, and equal amounts were resolved by SDS-PAGE, transferred onto nitrocellulose, and probed for the presence of phospho-MEK1/2, phospho-ERK1/2, total ERK1/2, total Rac1, p35, Cdk5, and RasGRF2 levels (panels are labeled for the detected proteins). Total Rac1 levels are similar in samples, whereas phospho-ERK1/2 levels are reduced after transfection of p35/Cdk5 (sample 3) (confirmed by the level of total ERK1/2 in this sample, which is higher than those of samples 2 and 4). Phospho-ERK1/2 levels are increased when the inactive Cdk5 is present (sample 4). The transfection of RasGRF2 causes an increase in phospho-ERK1/2 levels, whereas the transfection of p35/Cdk5 with RasGRF2 decreased this activation. The cotransfection of the inactive Cdk5 (lacking p35) causes the activated levels of ERK1/2 to return to the levels observed when only RasGRF2 was transfected. Immunodetected proteins are indicated next to the panels. D, Rac activity assays were performed to confirm that the serine 737 was indeed the phospho-site targeted by Cdk5 to mediate RasGRF2-mediated Rac activity. Tranfections are as follows: 1, EV; 2, RasGRF2; 3, RasGRF2+p35+Cdk5; 4, RasGRF2 S737A +p35+Cdk5; 5, RasGRF2 S717A +p35+Cdk5; 6, EV+GTP. The RasGRF2 S737A mutant behaved like wild-type RasGRF2; however, the RasGRF2 S717A mutant with p35 and Cdk5 showed the reduction in Rac activity demonstrating that serine 737 was the site targeted by Cdk5. E , Serine 737 phosphorylation by p35/Cdk5 reduces ERK1/2 activity. Transfections are as follows: 1, empty vector; 2, RasGRF2; 3, RasGRF2+p35+Cdk5; 4, RasGRF2 S737A +p35+Cdk5. Equal amounts were resolved by SDS-PAGE, transferred onto nitrocellulose, and probed for the presence of phospho-MEK1/2, phospho-ERK1/2, total ERK1/2, p35, Cdk5, and RasGRF2 levels. Immunodetected proteins are indicated next to the panels. The cotransfection of RasGRF2 S737A +p35+Cdk5 reversed the downregulation of ERK1/2 activity mediated by RasGRF2+p35+Cdk5, thus confirming that serine 737 is the site targeted by p35/Cdk5 to downregulate RasGRF2-mediated ERK1/2 activity.
    Figure Legend Snippet: p35/Cdk5 and RasGRF2 mediate cellular Rac activity and ERK1/2 activity. A , CHO cells were transfected with EV-GTP (lane 1), RasGRF2 (lane 2), RasGRF2+p35+Cdk5 (lane 3), RasGRF2+Cdk5 (lane4), and EV+GTP (lane5). CHO cell lysates were incubated with GST-PAK1 and GST beads for 1 hr. Beads with bound active Rac were washed three times in lysis–binding–wash buffer and eluted by addition of 2× SDS-sample buffer/β-mercaptoethanol and boiling for 5 min. Eluted, active Rac samples were resolved by SDS-PAGE, transferred onto nitrocellulose, and immunodetected using the monoclonal anti-Rac1 Ab provided in the kit at 1:1000 dilution. p35/Cdk5 reduced RasGRF2-mediated active Rac levels. EV lysate incubated with GTP before the pull-downs exhibited the highest active Rac levels (EV+GTP). Representative Western blots show active Rac1 levels and their corresponding total Rac levels. B , p35/Cdk5 does not affect RasGRF2-mediated Ras activity. Transfections are as follows: 1, EV; 2, RasGRF2; 3, RasGRF2+p35+Cdk5; 4, RasGRF2+Cdk5; 5, EV+GTP. CHO cell lysates were incubated with GST-Raf1 and GST beads for 1 hr. Beads with bound active Ras were washed three times before addition of 2× SDS-sample buffer/β-mercaptoethanol and boiling for 5 min. Eluted active Ras samples were resolved by SDS-PAGE and transferred onto nitrocellulose, and Ras was detected with the monoclonal anti-ras Ab provided in the kit. Immunodetected samples are indicated next to the panels. C , CHO cells were cotransfected with the following: 1, EV; 2, RasGRF2; 3, RasGRF2+p35+Cdk5; 4, RasGRF2+Cdk5. Lysates were harvested, and equal amounts were resolved by SDS-PAGE, transferred onto nitrocellulose, and probed for the presence of phospho-MEK1/2, phospho-ERK1/2, total ERK1/2, total Rac1, p35, Cdk5, and RasGRF2 levels (panels are labeled for the detected proteins). Total Rac1 levels are similar in samples, whereas phospho-ERK1/2 levels are reduced after transfection of p35/Cdk5 (sample 3) (confirmed by the level of total ERK1/2 in this sample, which is higher than those of samples 2 and 4). Phospho-ERK1/2 levels are increased when the inactive Cdk5 is present (sample 4). The transfection of RasGRF2 causes an increase in phospho-ERK1/2 levels, whereas the transfection of p35/Cdk5 with RasGRF2 decreased this activation. The cotransfection of the inactive Cdk5 (lacking p35) causes the activated levels of ERK1/2 to return to the levels observed when only RasGRF2 was transfected. Immunodetected proteins are indicated next to the panels. D, Rac activity assays were performed to confirm that the serine 737 was indeed the phospho-site targeted by Cdk5 to mediate RasGRF2-mediated Rac activity. Tranfections are as follows: 1, EV; 2, RasGRF2; 3, RasGRF2+p35+Cdk5; 4, RasGRF2 S737A +p35+Cdk5; 5, RasGRF2 S717A +p35+Cdk5; 6, EV+GTP. The RasGRF2 S737A mutant behaved like wild-type RasGRF2; however, the RasGRF2 S717A mutant with p35 and Cdk5 showed the reduction in Rac activity demonstrating that serine 737 was the site targeted by Cdk5. E , Serine 737 phosphorylation by p35/Cdk5 reduces ERK1/2 activity. Transfections are as follows: 1, empty vector; 2, RasGRF2; 3, RasGRF2+p35+Cdk5; 4, RasGRF2 S737A +p35+Cdk5. Equal amounts were resolved by SDS-PAGE, transferred onto nitrocellulose, and probed for the presence of phospho-MEK1/2, phospho-ERK1/2, total ERK1/2, p35, Cdk5, and RasGRF2 levels. Immunodetected proteins are indicated next to the panels. The cotransfection of RasGRF2 S737A +p35+Cdk5 reversed the downregulation of ERK1/2 activity mediated by RasGRF2+p35+Cdk5, thus confirming that serine 737 is the site targeted by p35/Cdk5 to downregulate RasGRF2-mediated ERK1/2 activity.

    Techniques Used: Activity Assay, Transfection, Incubation, Lysis, Binding Assay, SDS Page, Western Blot, Labeling, Activation Assay, Cotransfection, Mutagenesis, Plasmid Preparation

    12) Product Images from "Investigation of amino acid specificity in the CydX small protein shows sequence plasticity at the functional level"

    Article Title: Investigation of amino acid specificity in the CydX small protein shows sequence plasticity at the functional level

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0198699

    Sensitivity of alanine block CydX mutants to β-mercaptoethanol. A) Amino acid sequences of alanine block mutant CydX mutants. B) Zones of growth inhibition for wild-type, Δ cydX , and the three alanine block cydX mutants. The “Block 1” mutant contains alanines from positions 2 to 11, the “Block 2” contains alanines from positions 12–21, and the “Block 3” contains alanines from positions 22 to 31. All experiments were performed in at least triplicate, and the standard error of each experiment is shown.
    Figure Legend Snippet: Sensitivity of alanine block CydX mutants to β-mercaptoethanol. A) Amino acid sequences of alanine block mutant CydX mutants. B) Zones of growth inhibition for wild-type, Δ cydX , and the three alanine block cydX mutants. The “Block 1” mutant contains alanines from positions 2 to 11, the “Block 2” contains alanines from positions 12–21, and the “Block 3” contains alanines from positions 22 to 31. All experiments were performed in at least triplicate, and the standard error of each experiment is shown.

    Techniques Used: Blocking Assay, Mutagenesis, Inhibition

    Sensitivity of single amino acid CydX mutants to β-mercaptoethanol. A) Zones of growth inhibition for wild-type, Δ cydX and the cydX +Kan template strain used to make the mutants. B) Zones of growth inhibition for each single mutant strain. The E . coli CydX protein sequence is inset. All experiments were performed in at least triplicate, and the standard error of each experiment is shown.
    Figure Legend Snippet: Sensitivity of single amino acid CydX mutants to β-mercaptoethanol. A) Zones of growth inhibition for wild-type, Δ cydX and the cydX +Kan template strain used to make the mutants. B) Zones of growth inhibition for each single mutant strain. The E . coli CydX protein sequence is inset. All experiments were performed in at least triplicate, and the standard error of each experiment is shown.

    Techniques Used: Inhibition, Mutagenesis, Sequencing

    Sensitivity of double and triple amino acid CydX mutants to β-mercaptoethanol. A) Zones of growth inhibition for double and triple mutants. B) Growth of select double and triple cydX mutants in liquid culture containing β-mercaptoethanol. Samples are as follows: wild-type (filled circles), Δ cydX mutant (open circles), cydX+KAN (filled triangle), W2A/G9A mutant (open triangle), Y3A/G9A mutant (filled square), W6A/G9A mutant (open square), W2A/W6A/G9A mutant (closed diamond), Y3A/W6A/G9A mutant (open diamond), Y3A/G9A/L10G mutant (closed triangle). Strains with growth curves similar to wild-type are delineated by a dark line, strains with a growth phenotype similar to the deletion mutant are delineated with a dotted line, and strains with an intermediate growth phenotype are delineated by a grey line. Liquid culture experiments were conducted in Luria Broth containing 20mM β-mercaptoethanol. All experiments were performed in at least triplicate, and the standard error of each experiment is shown.
    Figure Legend Snippet: Sensitivity of double and triple amino acid CydX mutants to β-mercaptoethanol. A) Zones of growth inhibition for double and triple mutants. B) Growth of select double and triple cydX mutants in liquid culture containing β-mercaptoethanol. Samples are as follows: wild-type (filled circles), Δ cydX mutant (open circles), cydX+KAN (filled triangle), W2A/G9A mutant (open triangle), Y3A/G9A mutant (filled square), W6A/G9A mutant (open square), W2A/W6A/G9A mutant (closed diamond), Y3A/W6A/G9A mutant (open diamond), Y3A/G9A/L10G mutant (closed triangle). Strains with growth curves similar to wild-type are delineated by a dark line, strains with a growth phenotype similar to the deletion mutant are delineated with a dotted line, and strains with an intermediate growth phenotype are delineated by a grey line. Liquid culture experiments were conducted in Luria Broth containing 20mM β-mercaptoethanol. All experiments were performed in at least triplicate, and the standard error of each experiment is shown.

    Techniques Used: Inhibition, Mutagenesis

    13) Product Images from "The Interaction between the Drosophila Secreted Protein Argos and the Epidermal Growth Factor Receptor Inhibits Dimerization of the Receptor and Binding of Secreted Spitz to the Receptor"

    Article Title: The Interaction between the Drosophila Secreted Protein Argos and the Epidermal Growth Factor Receptor Inhibits Dimerization of the Receptor and Binding of Secreted Spitz to the Receptor

    Journal: Molecular and Cellular Biology

    doi:

    Aos, sSpi, and SpiAos bind to the DER extracellular domain. (A) DER/S2 cells incubated with Aos (lane 1), SpiAos (lane 2), or sSpi (lane 3) were treated with the cross-linker DTSSP, followed by immunoprecipitation (IP) by rat anti-DERc antibody. The cross-linker can be cleaved with 5% β-mercaptoethanol in Laemmli's sample buffer at 100°C for 5 min. Aos, SpiAos, and sSpi in the immunoprecipitates were detected by Western blotting analysis with anti-Myc or anti-Flag monoclonal antibodies. (B) Aos (10 nM) incubated with 0.01% BSA (lane 1) or 5 nM DER-Fc (lane 2), and subsequently with DTSSP, was precipitated by protein A and then probed with the anti-Aos monoclonal antibody. (C) sDER (20 nM), which had been incubated with 20 to 30 nM AosEGF-Fc (lane 1) or IgG1 Fc (lane 2) in the absence of the cross-linker, was precipitated with protein A beads and probed with mouse anti-sDER antibody. AosEGF-Fc and IgG1 Fc in the precipitates were detected by anti-Fc antibody. Experiments were performed twice with similar results.
    Figure Legend Snippet: Aos, sSpi, and SpiAos bind to the DER extracellular domain. (A) DER/S2 cells incubated with Aos (lane 1), SpiAos (lane 2), or sSpi (lane 3) were treated with the cross-linker DTSSP, followed by immunoprecipitation (IP) by rat anti-DERc antibody. The cross-linker can be cleaved with 5% β-mercaptoethanol in Laemmli's sample buffer at 100°C for 5 min. Aos, SpiAos, and sSpi in the immunoprecipitates were detected by Western blotting analysis with anti-Myc or anti-Flag monoclonal antibodies. (B) Aos (10 nM) incubated with 0.01% BSA (lane 1) or 5 nM DER-Fc (lane 2), and subsequently with DTSSP, was precipitated by protein A and then probed with the anti-Aos monoclonal antibody. (C) sDER (20 nM), which had been incubated with 20 to 30 nM AosEGF-Fc (lane 1) or IgG1 Fc (lane 2) in the absence of the cross-linker, was precipitated with protein A beads and probed with mouse anti-sDER antibody. AosEGF-Fc and IgG1 Fc in the precipitates were detected by anti-Fc antibody. Experiments were performed twice with similar results.

    Techniques Used: Incubation, Immunoprecipitation, Western Blot

    14) Product Images from "The R163K Mutant of Human Thymidylate Synthase Is Stabilized in an Active Conformation: Structural Asymmetry and Reactivity of Cysteine 195"

    Article Title: The R163K Mutant of Human Thymidylate Synthase Is Stabilized in an Active Conformation: Structural Asymmetry and Reactivity of Cysteine 195

    Journal: Biochemistry

    doi: 10.1021/bi7019386

    Stereoview of the active sites of the dimer in crystal form 2 with 2 F o - F c electron density, contoured at the 1.0σ level, for subunits A (top) and B (middle). Superposition of subunits A (in yellow) and B (in blue) is shown in the bottom panel. The β-mercaptoethanol molecule is connected to the side chain of Cys195 by a disulfide bond in subunit B while it forms a noncovalent adduct in subunit A. The distances from Cys195 to Ser216 are also different. Different oxidation states of Cys195 reflect its propensity to oxidation.
    Figure Legend Snippet: Stereoview of the active sites of the dimer in crystal form 2 with 2 F o - F c electron density, contoured at the 1.0σ level, for subunits A (top) and B (middle). Superposition of subunits A (in yellow) and B (in blue) is shown in the bottom panel. The β-mercaptoethanol molecule is connected to the side chain of Cys195 by a disulfide bond in subunit B while it forms a noncovalent adduct in subunit A. The distances from Cys195 to Ser216 are also different. Different oxidation states of Cys195 reflect its propensity to oxidation.

    Techniques Used:

    15) Product Images from "Conservation analysis of the CydX protein yields insights into small protein identification and evolution"

    Article Title: Conservation analysis of the CydX protein yields insights into small protein identification and evolution

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-15-946

    Confirmation of functionality of CydX homologues. (A) Alignment of protein sequences of CydX homologues from Escherichia coli and other bacteria species. The small protein from Burkholderia sp. 383 (“Burkholderia383”) is not thought to be a homologue and was included as a negative control for the assay. Based on its significant sequence divergence was included in a separate alignment. (B) Alignment of the E. coli CydX protein with the CydZ protein from Klebsiella pneumoniae . (C) Assay of complementation of the Δ cydX β-mercaptoethanol sensitivity phenotype by expression of potential CydX homologues, a false positive from the tblastn search ( Burkholderia sp. 383 ), and an unrelated small protein (CydZ) from a different bacterial species. Sensitivity was measured using zones of inhibition, and the diameter of the zone after addition of 10 μL of 12 M β-mercaptoethanol to a plate of bacteria is shown. Species are as follows: Escherichia coli (“Escherichia”), Pectobacterium atrosepticus (“Pectobacterium”), Burkholderia xenovorans (“Burkholderia”), Actinobacillus pleuropneumoniae (“Actinobacillus”), Burkholderia sp. 383 (“Burkholderia sp. 383”), Klebsiella pneumoniae (“Klebsiella”), Cellvibrio japonicus Ueda107 (“Cellvibrio”), Methylibium petroleiphilum PM1 (“Methylibium”), Haemophilus influenzae 10810 (“Haemophilus”), and Francisella philomiragia subsp. Philomiragia ATCC 25017 (“Francisella”). Alignments were generated using the program MUSCLE [ 57 ]. Amino acids are colored based on their properties at physiological conditions as follows: red amino acids are hydrophobic, green residues are hydrophilic, purple residues are positively-charged and blue residues are negatively-charged. ‘*’ indicates that the residues are identical in all sequences and ‘:’ and ‘.’, respectively, indicated conserved and semi-conserved substitutions as defined by MUSCLE.
    Figure Legend Snippet: Confirmation of functionality of CydX homologues. (A) Alignment of protein sequences of CydX homologues from Escherichia coli and other bacteria species. The small protein from Burkholderia sp. 383 (“Burkholderia383”) is not thought to be a homologue and was included as a negative control for the assay. Based on its significant sequence divergence was included in a separate alignment. (B) Alignment of the E. coli CydX protein with the CydZ protein from Klebsiella pneumoniae . (C) Assay of complementation of the Δ cydX β-mercaptoethanol sensitivity phenotype by expression of potential CydX homologues, a false positive from the tblastn search ( Burkholderia sp. 383 ), and an unrelated small protein (CydZ) from a different bacterial species. Sensitivity was measured using zones of inhibition, and the diameter of the zone after addition of 10 μL of 12 M β-mercaptoethanol to a plate of bacteria is shown. Species are as follows: Escherichia coli (“Escherichia”), Pectobacterium atrosepticus (“Pectobacterium”), Burkholderia xenovorans (“Burkholderia”), Actinobacillus pleuropneumoniae (“Actinobacillus”), Burkholderia sp. 383 (“Burkholderia sp. 383”), Klebsiella pneumoniae (“Klebsiella”), Cellvibrio japonicus Ueda107 (“Cellvibrio”), Methylibium petroleiphilum PM1 (“Methylibium”), Haemophilus influenzae 10810 (“Haemophilus”), and Francisella philomiragia subsp. Philomiragia ATCC 25017 (“Francisella”). Alignments were generated using the program MUSCLE [ 57 ]. Amino acids are colored based on their properties at physiological conditions as follows: red amino acids are hydrophobic, green residues are hydrophilic, purple residues are positively-charged and blue residues are negatively-charged. ‘*’ indicates that the residues are identical in all sequences and ‘:’ and ‘.’, respectively, indicated conserved and semi-conserved substitutions as defined by MUSCLE.

    Techniques Used: Negative Control, Sequencing, Expressing, Inhibition, Generated

    Testing the functional importance of the CydX C-terminal amino acids. (A) Alignment of the E. coli CydX protein sequence along with six mutant sequences containing mutated C-terminal amino acid sequences. (B) Assay of CydX function was conducted using a zone assay testing the sensitivity to β-mercaptoethanol. Sensitivity was measured using zones of inhibition, and the diameter of the zone after addition of 10 μL of 12 M β-mercaptoethanol to a plate of bacteria is shown. The average and standard deviation of zone sizes was calculated from at least three replicate plates. Alignments were generated using the program MUSCLE [ 57 ].
    Figure Legend Snippet: Testing the functional importance of the CydX C-terminal amino acids. (A) Alignment of the E. coli CydX protein sequence along with six mutant sequences containing mutated C-terminal amino acid sequences. (B) Assay of CydX function was conducted using a zone assay testing the sensitivity to β-mercaptoethanol. Sensitivity was measured using zones of inhibition, and the diameter of the zone after addition of 10 μL of 12 M β-mercaptoethanol to a plate of bacteria is shown. The average and standard deviation of zone sizes was calculated from at least three replicate plates. Alignments were generated using the program MUSCLE [ 57 ].

    Techniques Used: Functional Assay, Sequencing, Mutagenesis, Inhibition, Standard Deviation, Generated

    16) Product Images from "Structural and Functional Characterization of Factor H Mutations Associated with Atypical Hemolytic Uremic Syndrome"

    Article Title: Structural and Functional Characterization of Factor H Mutations Associated with Atypical Hemolytic Uremic Syndrome

    Journal: American Journal of Human Genetics

    doi:

    Characterization of the high-molecular-weight factor H associated with the R1210C mutation. a, 10% SDS-PAGE of the factor H (fH) proteins purified from patient HUS29 and control individual N3. Samples were reduced with 10% β-mercaptoethanol. A gel stained with 1% Coomassie is shown. b, Western blot analyses of reduced and nonreduced factor H proteins purified from patient HUS29 and control individual N3, using a rabbit anti-HSA polyclonal antibody. c, SDS-PAGE analysis of the wild-type (H29R; flow through) and mutated (H29C; retained) factor H proteins purified from patient HUS29, using anti-HSA affinity chromatography. Samples were not reduced. A silver-stained 10% polyacrylamide gel is shown.
    Figure Legend Snippet: Characterization of the high-molecular-weight factor H associated with the R1210C mutation. a, 10% SDS-PAGE of the factor H (fH) proteins purified from patient HUS29 and control individual N3. Samples were reduced with 10% β-mercaptoethanol. A gel stained with 1% Coomassie is shown. b, Western blot analyses of reduced and nonreduced factor H proteins purified from patient HUS29 and control individual N3, using a rabbit anti-HSA polyclonal antibody. c, SDS-PAGE analysis of the wild-type (H29R; flow through) and mutated (H29C; retained) factor H proteins purified from patient HUS29, using anti-HSA affinity chromatography. Samples were not reduced. A silver-stained 10% polyacrylamide gel is shown.

    Techniques Used: Molecular Weight, Mutagenesis, SDS Page, Purification, Staining, Western Blot, Flow Cytometry, Affinity Chromatography

    17) Product Images from "Investigation of amino acid specificity in the CydX small protein shows sequence plasticity at the functional level"

    Article Title: Investigation of amino acid specificity in the CydX small protein shows sequence plasticity at the functional level

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0198699

    Sensitivity of alanine block CydX mutants to β-mercaptoethanol. A) Amino acid sequences of alanine block mutant CydX mutants. B) Zones of growth inhibition for wild-type, Δ cydX , and the three alanine block cydX mutants. The “Block 1” mutant contains alanines from positions 2 to 11, the “Block 2” contains alanines from positions 12–21, and the “Block 3” contains alanines from positions 22 to 31. All experiments were performed in at least triplicate, and the standard error of each experiment is shown.
    Figure Legend Snippet: Sensitivity of alanine block CydX mutants to β-mercaptoethanol. A) Amino acid sequences of alanine block mutant CydX mutants. B) Zones of growth inhibition for wild-type, Δ cydX , and the three alanine block cydX mutants. The “Block 1” mutant contains alanines from positions 2 to 11, the “Block 2” contains alanines from positions 12–21, and the “Block 3” contains alanines from positions 22 to 31. All experiments were performed in at least triplicate, and the standard error of each experiment is shown.

    Techniques Used: Blocking Assay, Mutagenesis, Inhibition

    Sensitivity of single amino acid CydX mutants to β-mercaptoethanol. A) Zones of growth inhibition for wild-type, Δ cydX and the cydX +Kan template strain used to make the mutants. B) Zones of growth inhibition for each single mutant strain. The E . coli CydX protein sequence is inset. All experiments were performed in at least triplicate, and the standard error of each experiment is shown.
    Figure Legend Snippet: Sensitivity of single amino acid CydX mutants to β-mercaptoethanol. A) Zones of growth inhibition for wild-type, Δ cydX and the cydX +Kan template strain used to make the mutants. B) Zones of growth inhibition for each single mutant strain. The E . coli CydX protein sequence is inset. All experiments were performed in at least triplicate, and the standard error of each experiment is shown.

    Techniques Used: Inhibition, Mutagenesis, Sequencing

    Sensitivity of double and triple amino acid CydX mutants to β-mercaptoethanol. A) Zones of growth inhibition for double and triple mutants. B) Growth of select double and triple cydX mutants in liquid culture containing β-mercaptoethanol. Samples are as follows: wild-type (filled circles), Δ cydX mutant (open circles), cydX+KAN (filled triangle), W2A/G9A mutant (open triangle), Y3A/G9A mutant (filled square), W6A/G9A mutant (open square), W2A/W6A/G9A mutant (closed diamond), Y3A/W6A/G9A mutant (open diamond), Y3A/G9A/L10G mutant (closed triangle). Strains with growth curves similar to wild-type are delineated by a dark line, strains with a growth phenotype similar to the deletion mutant are delineated with a dotted line, and strains with an intermediate growth phenotype are delineated by a grey line. Liquid culture experiments were conducted in Luria Broth containing 20mM β-mercaptoethanol. All experiments were performed in at least triplicate, and the standard error of each experiment is shown.
    Figure Legend Snippet: Sensitivity of double and triple amino acid CydX mutants to β-mercaptoethanol. A) Zones of growth inhibition for double and triple mutants. B) Growth of select double and triple cydX mutants in liquid culture containing β-mercaptoethanol. Samples are as follows: wild-type (filled circles), Δ cydX mutant (open circles), cydX+KAN (filled triangle), W2A/G9A mutant (open triangle), Y3A/G9A mutant (filled square), W6A/G9A mutant (open square), W2A/W6A/G9A mutant (closed diamond), Y3A/W6A/G9A mutant (open diamond), Y3A/G9A/L10G mutant (closed triangle). Strains with growth curves similar to wild-type are delineated by a dark line, strains with a growth phenotype similar to the deletion mutant are delineated with a dotted line, and strains with an intermediate growth phenotype are delineated by a grey line. Liquid culture experiments were conducted in Luria Broth containing 20mM β-mercaptoethanol. All experiments were performed in at least triplicate, and the standard error of each experiment is shown.

    Techniques Used: Inhibition, Mutagenesis

    18) Product Images from "Bacterial protein translocation requires only one copy of the SecY complex in vivo"

    Article Title: Bacterial protein translocation requires only one copy of the SecY complex in vivo

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201205140

    Saturation of SecY channels with a post-translational translocation intermediate. (A) Scheme of a post-translational translocation intermediate generated with SecA and the substrate OmpA-GFP. The translocating chain contains the signal sequence of OmpA at the N terminus and the “superfolder” GFP at the C terminus. Its insertion into the SecY channel is monitored by disulfide bridge formation between cysteines in the substrate and SecY. (B) The insertion of OmpA-GFP, containing a cysteine at position 21 (21C), into SecY containing a cysteine at position 68 (68C) was tested by disulfide bridge formation after the addition of the oxidant CuPh 3 to intact E. coli cells. Where indicated, the substrate or SecY lacked a cysteine or the cysteines were blocked with N -ethylmaleimide (NEM) before addition of CuPh 3 . As a control, a substrate was used with a defective signal sequence (RR-21C). Where indicated, disulfide bridges were reduced by β-mercaptoethanol (β-ME). All samples were analyzed by SDS-PAGE followed by blotting with SecY or GFP antibodies. (C) OmpA-GFP was expressed from the arabinose (Ara)-inducible promoter in cells producing SecY at approximately endogenous level using a GUG translational start codon (plasmid pACYC-SecYEG). After addition of CuPh 3 , the lysate was analyzed by SDS-PAGE and blotting with SecY antibodies. Where indicated, rifampicin (Rif) was added for different time periods. Red arrows and black asterisks indicate cross-links between SecY and substrate or endogenous proteins, respectively. The right panel shows quantification of the cross-linking efficiency between SecY and translocation substrates, based on the decrease of noncross-linked SecY in experiments such as shown in the left panel and Fig. 2 B . Three different experiments were analyzed (mean and SD). (D) OmpA-GFP with either a wild-type (WT) or defective (RR) signal sequence was expressed under the arabinose (Ara) promoter in cells producing wild-type SecY or SecY lacking its plug domain (ΔP). Controls were performed with an empty vector (vec) and without Ara induction. Biotin-maleimide was added to the cells, and the modification of proteins was probed by SDS-PAGE and blotting with HRP-conjugated streptavidin. The samples were also probed with SecY, GFP, and trigger factor (TF; loading control) antibodies. Where indicated, cells were pretreated with rifampicin (Rif) before addition of biotin-maleimide. p30, a prominently modified cytosolic protein. The blue arrowheads indicate biotinylation of translocation-incompetent OmpA-GFP carrying a defective signal sequence.
    Figure Legend Snippet: Saturation of SecY channels with a post-translational translocation intermediate. (A) Scheme of a post-translational translocation intermediate generated with SecA and the substrate OmpA-GFP. The translocating chain contains the signal sequence of OmpA at the N terminus and the “superfolder” GFP at the C terminus. Its insertion into the SecY channel is monitored by disulfide bridge formation between cysteines in the substrate and SecY. (B) The insertion of OmpA-GFP, containing a cysteine at position 21 (21C), into SecY containing a cysteine at position 68 (68C) was tested by disulfide bridge formation after the addition of the oxidant CuPh 3 to intact E. coli cells. Where indicated, the substrate or SecY lacked a cysteine or the cysteines were blocked with N -ethylmaleimide (NEM) before addition of CuPh 3 . As a control, a substrate was used with a defective signal sequence (RR-21C). Where indicated, disulfide bridges were reduced by β-mercaptoethanol (β-ME). All samples were analyzed by SDS-PAGE followed by blotting with SecY or GFP antibodies. (C) OmpA-GFP was expressed from the arabinose (Ara)-inducible promoter in cells producing SecY at approximately endogenous level using a GUG translational start codon (plasmid pACYC-SecYEG). After addition of CuPh 3 , the lysate was analyzed by SDS-PAGE and blotting with SecY antibodies. Where indicated, rifampicin (Rif) was added for different time periods. Red arrows and black asterisks indicate cross-links between SecY and substrate or endogenous proteins, respectively. The right panel shows quantification of the cross-linking efficiency between SecY and translocation substrates, based on the decrease of noncross-linked SecY in experiments such as shown in the left panel and Fig. 2 B . Three different experiments were analyzed (mean and SD). (D) OmpA-GFP with either a wild-type (WT) or defective (RR) signal sequence was expressed under the arabinose (Ara) promoter in cells producing wild-type SecY or SecY lacking its plug domain (ΔP). Controls were performed with an empty vector (vec) and without Ara induction. Biotin-maleimide was added to the cells, and the modification of proteins was probed by SDS-PAGE and blotting with HRP-conjugated streptavidin. The samples were also probed with SecY, GFP, and trigger factor (TF; loading control) antibodies. Where indicated, cells were pretreated with rifampicin (Rif) before addition of biotin-maleimide. p30, a prominently modified cytosolic protein. The blue arrowheads indicate biotinylation of translocation-incompetent OmpA-GFP carrying a defective signal sequence.

    Techniques Used: Translocation Assay, Generated, Sequencing, SDS Page, Acetylene Reduction Assay, Plasmid Preparation, Modification

    SecY complexes interact in vivo through different surfaces. (A) View of Thermotoga maritima SecY complex from the periplasm. The N- and C-terminal halves of SecY are colored blue and red, respectively, SecG in green, and SecE in yellow. Balls in magenta indicate positions that were mutated in the E. coli protein to cysteines. Note that two cysteines are part of an inserted segment. (B) The accessibility of cysteine residues introduced into SecY was tested in intact E. coli by modification with biotin-PEG 2 -maleimide, followed by incubation with streptavidin (SA). The samples were analyzed by SDS-PAGE and immunoblotting with SecY antibodies (anti-SecY). The two bands correspond to one or two SecY molecules bound to tetrameric streptavidin. (C) The interaction of SecYs with the indicated cysteines was tested by addition of bismaleimide-PEG 3 (BM-PEG 3 ) to resuspended intact E. coli cells, pretreated with rifampicin for 30 min. The samples were analyzed by SDS-PAGE and immunoblotting for SecY. SecY 2 , cross-linked SecY dimers. (D) The front-to-front interaction of SecYs with the indicated cysteines was tested by spontaneous disulfide bridge formation in vivo. Where indicated, the samples were treated with β-mercaptoethanol (β-ME). (E) Scheme of a SecY complex containing SecE, SecG, and SecY in a single polypeptide chain. The gray dotted segments are added linkers. Residue L106 at the back of SecE (see A) is indicated. (F) The back-to-back interaction of single-chain SecY complexes with a cysteine at position 106 of SecE was tested by disulfide bridge formation after addition of the oxidant CuPh 3 to cell lysates. Controls were performed with protein lacking a cysteine and with β-ME addition after cross-linking.
    Figure Legend Snippet: SecY complexes interact in vivo through different surfaces. (A) View of Thermotoga maritima SecY complex from the periplasm. The N- and C-terminal halves of SecY are colored blue and red, respectively, SecG in green, and SecE in yellow. Balls in magenta indicate positions that were mutated in the E. coli protein to cysteines. Note that two cysteines are part of an inserted segment. (B) The accessibility of cysteine residues introduced into SecY was tested in intact E. coli by modification with biotin-PEG 2 -maleimide, followed by incubation with streptavidin (SA). The samples were analyzed by SDS-PAGE and immunoblotting with SecY antibodies (anti-SecY). The two bands correspond to one or two SecY molecules bound to tetrameric streptavidin. (C) The interaction of SecYs with the indicated cysteines was tested by addition of bismaleimide-PEG 3 (BM-PEG 3 ) to resuspended intact E. coli cells, pretreated with rifampicin for 30 min. The samples were analyzed by SDS-PAGE and immunoblotting for SecY. SecY 2 , cross-linked SecY dimers. (D) The front-to-front interaction of SecYs with the indicated cysteines was tested by spontaneous disulfide bridge formation in vivo. Where indicated, the samples were treated with β-mercaptoethanol (β-ME). (E) Scheme of a SecY complex containing SecE, SecG, and SecY in a single polypeptide chain. The gray dotted segments are added linkers. Residue L106 at the back of SecE (see A) is indicated. (F) The back-to-back interaction of single-chain SecY complexes with a cysteine at position 106 of SecE was tested by disulfide bridge formation after addition of the oxidant CuPh 3 to cell lysates. Controls were performed with protein lacking a cysteine and with β-ME addition after cross-linking.

    Techniques Used: In Vivo, Modification, Incubation, SDS Page

    19) Product Images from "Equal Neutralization Potency of Antibodies Raised against Abrin Subunits"

    Article Title: Equal Neutralization Potency of Antibodies Raised against Abrin Subunits

    Journal: Antibodies

    doi: 10.3390/antib9010004

    Gel electrophoresis of chimera abrin/ricin toxins. SDS-PAGE of: Reconstituted abrin (lane 1), Reconstituted ricin (lane 2), Chimera toxin A ricin B abrin (lane 3), and Chimera toxin A abrin B ricin (lane 4) was performed ( A ) in the absence of reducing agent, or ( B ) in the presence of β-mercaptoethanol.
    Figure Legend Snippet: Gel electrophoresis of chimera abrin/ricin toxins. SDS-PAGE of: Reconstituted abrin (lane 1), Reconstituted ricin (lane 2), Chimera toxin A ricin B abrin (lane 3), and Chimera toxin A abrin B ricin (lane 4) was performed ( A ) in the absence of reducing agent, or ( B ) in the presence of β-mercaptoethanol.

    Techniques Used: Nucleic Acid Electrophoresis, SDS Page

    20) Product Images from "A Novel Conserved Isoform of the Ubiquitin Ligase UFD2a/UBE4B Is Expressed Exclusively in Mature Striated Muscle Cells"

    Article Title: A Novel Conserved Isoform of the Ubiquitin Ligase UFD2a/UBE4B Is Expressed Exclusively in Mature Striated Muscle Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0028861

    The muscle-specific UFD2a-7/7a isoform does not interact with VCP/p97. A). Yeast cells transformed with bait plasmids pDEST32-UFD2a, pDEST32-UFD2a-7/7a, or empty pDEST32 and prey plasmids pDEST22-VCP/p97 or empty pDEST22 were grown in liquid double dropout medium (-leu, -trp) to saturation and diluted to an OD 600 of 1. Serial dilutions of these cell cultures were prepared in 96-well plates and transferred to triple-dropout agar plates (-leu, -trp, -his) supplemented with 25 mM 3-AT. B). Isolated colonies of yeast cells transformed (as in panel A) were grown to saturation, diluted to OD 600 of 0.3, and grown to log phase (OD 600 = 0.8-1.5). Cells were disrupted with glass beads in Z buffer; ONPG substrate and β-mercaptoethanol were added to the resulting supernatants. After incubation at 37°C, supernatants were transferred to 96-well plates and read at 420 nm. B). Relative β-galactosidase activity is calculated from 6 independent experiments; error bars indicate standard error. The average activity of UFD2a+VCP/p97 was significantly higher than that of the other transformants (*p = 0.02). The activity of UFD2a-7/7a+VCP/p97 did not differ from that of the negative controls.
    Figure Legend Snippet: The muscle-specific UFD2a-7/7a isoform does not interact with VCP/p97. A). Yeast cells transformed with bait plasmids pDEST32-UFD2a, pDEST32-UFD2a-7/7a, or empty pDEST32 and prey plasmids pDEST22-VCP/p97 or empty pDEST22 were grown in liquid double dropout medium (-leu, -trp) to saturation and diluted to an OD 600 of 1. Serial dilutions of these cell cultures were prepared in 96-well plates and transferred to triple-dropout agar plates (-leu, -trp, -his) supplemented with 25 mM 3-AT. B). Isolated colonies of yeast cells transformed (as in panel A) were grown to saturation, diluted to OD 600 of 0.3, and grown to log phase (OD 600 = 0.8-1.5). Cells were disrupted with glass beads in Z buffer; ONPG substrate and β-mercaptoethanol were added to the resulting supernatants. After incubation at 37°C, supernatants were transferred to 96-well plates and read at 420 nm. B). Relative β-galactosidase activity is calculated from 6 independent experiments; error bars indicate standard error. The average activity of UFD2a+VCP/p97 was significantly higher than that of the other transformants (*p = 0.02). The activity of UFD2a-7/7a+VCP/p97 did not differ from that of the negative controls.

    Techniques Used: Transformation Assay, Isolation, Incubation, Activity Assay

    21) Product Images from "Mechanism-Based Inactivation of Human Cytochrome P450 2B6 by Chlorpyrifos"

    Article Title: Mechanism-Based Inactivation of Human Cytochrome P450 2B6 by Chlorpyrifos

    Journal: Chemical research in toxicology

    doi: 10.1021/acs.chemrestox.5b00156

    SDS–PAGE analysis of CYP2B6 after inactivation by chlorpyrifos. CYP2B6 was inactivated at 37 °C for 10 min in the presence of 100 μ M chlorpyrifos as described in Materials and Methods. Following inactivation, the proteins were loaded onto a 10% polyacrylamide gel and separated by SDS–PAGE and stained with Coomassie blue for visualization. Lane 1, CYP2B6 minus NADPH. Lane 2, CYP2B6 plus NADPH. Lane 3, CYP2B6 − NADPH + 3.92 mM β -mercaptoethanol. Lane 4, CYP2B6 + NADPH + 3.92 mM β -mercaptoethanol.
    Figure Legend Snippet: SDS–PAGE analysis of CYP2B6 after inactivation by chlorpyrifos. CYP2B6 was inactivated at 37 °C for 10 min in the presence of 100 μ M chlorpyrifos as described in Materials and Methods. Following inactivation, the proteins were loaded onto a 10% polyacrylamide gel and separated by SDS–PAGE and stained with Coomassie blue for visualization. Lane 1, CYP2B6 minus NADPH. Lane 2, CYP2B6 plus NADPH. Lane 3, CYP2B6 − NADPH + 3.92 mM β -mercaptoethanol. Lane 4, CYP2B6 + NADPH + 3.92 mM β -mercaptoethanol.

    Techniques Used: SDS Page, Staining

    22) Product Images from "Investigation of amino acid specificity in the CydX small protein shows sequence plasticity at the functional level"

    Article Title: Investigation of amino acid specificity in the CydX small protein shows sequence plasticity at the functional level

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0198699

    Sensitivity of alanine block CydX mutants to β-mercaptoethanol. A) Amino acid sequences of alanine block mutant CydX mutants. B) Zones of growth inhibition for wild-type, Δ cydX , and the three alanine block cydX mutants. The “Block 1” mutant contains alanines from positions 2 to 11, the “Block 2” contains alanines from positions 12–21, and the “Block 3” contains alanines from positions 22 to 31. All experiments were performed in at least triplicate, and the standard error of each experiment is shown.
    Figure Legend Snippet: Sensitivity of alanine block CydX mutants to β-mercaptoethanol. A) Amino acid sequences of alanine block mutant CydX mutants. B) Zones of growth inhibition for wild-type, Δ cydX , and the three alanine block cydX mutants. The “Block 1” mutant contains alanines from positions 2 to 11, the “Block 2” contains alanines from positions 12–21, and the “Block 3” contains alanines from positions 22 to 31. All experiments were performed in at least triplicate, and the standard error of each experiment is shown.

    Techniques Used: Blocking Assay, Mutagenesis, Inhibition

    Sensitivity of single amino acid CydX mutants to β-mercaptoethanol. A) Zones of growth inhibition for wild-type, Δ cydX and the cydX +Kan template strain used to make the mutants. B) Zones of growth inhibition for each single mutant strain. The E . coli CydX protein sequence is inset. All experiments were performed in at least triplicate, and the standard error of each experiment is shown.
    Figure Legend Snippet: Sensitivity of single amino acid CydX mutants to β-mercaptoethanol. A) Zones of growth inhibition for wild-type, Δ cydX and the cydX +Kan template strain used to make the mutants. B) Zones of growth inhibition for each single mutant strain. The E . coli CydX protein sequence is inset. All experiments were performed in at least triplicate, and the standard error of each experiment is shown.

    Techniques Used: Inhibition, Mutagenesis, Sequencing

    Sensitivity of double and triple amino acid CydX mutants to β-mercaptoethanol. A) Zones of growth inhibition for double and triple mutants. B) Growth of select double and triple cydX mutants in liquid culture containing β-mercaptoethanol. Samples are as follows: wild-type (filled circles), Δ cydX mutant (open circles), cydX+KAN (filled triangle), W2A/G9A mutant (open triangle), Y3A/G9A mutant (filled square), W6A/G9A mutant (open square), W2A/W6A/G9A mutant (closed diamond), Y3A/W6A/G9A mutant (open diamond), Y3A/G9A/L10G mutant (closed triangle). Strains with growth curves similar to wild-type are delineated by a dark line, strains with a growth phenotype similar to the deletion mutant are delineated with a dotted line, and strains with an intermediate growth phenotype are delineated by a grey line. Liquid culture experiments were conducted in Luria Broth containing 20mM β-mercaptoethanol. All experiments were performed in at least triplicate, and the standard error of each experiment is shown.
    Figure Legend Snippet: Sensitivity of double and triple amino acid CydX mutants to β-mercaptoethanol. A) Zones of growth inhibition for double and triple mutants. B) Growth of select double and triple cydX mutants in liquid culture containing β-mercaptoethanol. Samples are as follows: wild-type (filled circles), Δ cydX mutant (open circles), cydX+KAN (filled triangle), W2A/G9A mutant (open triangle), Y3A/G9A mutant (filled square), W6A/G9A mutant (open square), W2A/W6A/G9A mutant (closed diamond), Y3A/W6A/G9A mutant (open diamond), Y3A/G9A/L10G mutant (closed triangle). Strains with growth curves similar to wild-type are delineated by a dark line, strains with a growth phenotype similar to the deletion mutant are delineated with a dotted line, and strains with an intermediate growth phenotype are delineated by a grey line. Liquid culture experiments were conducted in Luria Broth containing 20mM β-mercaptoethanol. All experiments were performed in at least triplicate, and the standard error of each experiment is shown.

    Techniques Used: Inhibition, Mutagenesis

    23) Product Images from "Metabolic and thermal stimuli control K2P2.1 (TREK-1) through modular sensory and gating domains"

    Article Title: Metabolic and thermal stimuli control K2P2.1 (TREK-1) through modular sensory and gating domains

    Journal: The EMBO Journal

    doi: 10.1038/emboj.2012.171

    Ct domains act cooperatively to affect K 2P 2.1 (TREK-1) function. ( A ) Immunoblot analysis of lysates from oocytes expressing HA-tagged WT, HA-WT or tandem HA-WT-WT K 2P 2.1 (TREK-1) channels. Lysates were pre-incubated with or without EndoH for 1 h at 4 o C or at room temperature (RT). Before electrophoresis, all samples were treated with 2% SDS and 2% β-mercaptoethanol for 15 min at 50 o C to dissociate K 2P 2.1 (TREK-1) subunits. Asterisks denote deglycolyslated forms of WT and tandem channels. ( B–D ) Normalized responses of the indicated K 2P 2.1 (TREK-1) channels to pH o changes 2 mM [K + ] o . Currents were elicited by a voltage ramp from −150 to +50 mV, from a holding potential of −80 mV. Data (mean±s.e., n ⩾6, N ⩾2) was taken at 0 mV, normalized to activity at pH 9.0 and fitted to the Hill equation.
    Figure Legend Snippet: Ct domains act cooperatively to affect K 2P 2.1 (TREK-1) function. ( A ) Immunoblot analysis of lysates from oocytes expressing HA-tagged WT, HA-WT or tandem HA-WT-WT K 2P 2.1 (TREK-1) channels. Lysates were pre-incubated with or without EndoH for 1 h at 4 o C or at room temperature (RT). Before electrophoresis, all samples were treated with 2% SDS and 2% β-mercaptoethanol for 15 min at 50 o C to dissociate K 2P 2.1 (TREK-1) subunits. Asterisks denote deglycolyslated forms of WT and tandem channels. ( B–D ) Normalized responses of the indicated K 2P 2.1 (TREK-1) channels to pH o changes 2 mM [K + ] o . Currents were elicited by a voltage ramp from −150 to +50 mV, from a holding potential of −80 mV. Data (mean±s.e., n ⩾6, N ⩾2) was taken at 0 mV, normalized to activity at pH 9.0 and fitted to the Hill equation.

    Techniques Used: Activated Clotting Time Assay, Expressing, Incubation, Electrophoresis, Activity Assay

    24) Product Images from "The Protein Kinase A Pathway Contributes to Hg2+-Induced Alterations in Phosphorylation and Subcellular Distribution of Occludin Associated with Increased Tight Junction Permeability of Salivary Epithelial Cell Monolayers"

    Article Title: The Protein Kinase A Pathway Contributes to Hg2+-Induced Alterations in Phosphorylation and Subcellular Distribution of Occludin Associated with Increased Tight Junction Permeability of Salivary Epithelial Cell Monolayers

    Journal:

    doi: 10.1124/jpet.107.135798

    Hg 2+ -induced increase in the tight junction permeability is independent of its cysteine cross-linking property. Cells were treated with the reducing agent (cysteine crosslinking inhibitor) β-mercaptoethanol at time 0, and mercuric chloride was
    Figure Legend Snippet: Hg 2+ -induced increase in the tight junction permeability is independent of its cysteine cross-linking property. Cells were treated with the reducing agent (cysteine crosslinking inhibitor) β-mercaptoethanol at time 0, and mercuric chloride was

    Techniques Used: Permeability

    25) Product Images from "Oxidation Resistance 1 Modulates Glycolytic Pathways in the Cerebellum via an Interaction with Glucose-6-Phosphate Isomerase"

    Article Title: Oxidation Resistance 1 Modulates Glycolytic Pathways in the Cerebellum via an Interaction with Glucose-6-Phosphate Isomerase

    Journal: Molecular Neurobiology

    doi: 10.1007/s12035-018-1174-x

    Oxr1 modulates Gpi1 oligomerisation. a Dimerisation of Gpi1 in cells co-transfected with Gpi1 and either an empty vector or full-length (Oxr1-FL) or short (Oxr1-C) Oxr1 isoforms. Cells were treated with a cross-linker (DSP) and proteins were extracted in PBS; the loading buffer did not contain any reducing β-mercaptoethanol and samples were not boiled (non-reducing conditions). As a control, protein extracts from cells treated with DSP were incubated with the reducing agent β-mercaptoethanol and boiled (DSPβ/b). Vinculin (Vin) levels were used to control for equivalent loading. b – c Quantification of the dimeric ( b ) or tetrameric ( c ) versus monomeric forms of Gpi1 ( N = 6 independent repeats). d – e Western blot and quantification showing Gpi1 oligomerization in cerebellum from Oxr1 d/d and Oxr1 +/+ mice from proteins extracted in PBS and non-reducing conditions. Ponceau staining was used to control for equal loading. As a control, protein extracts from the same preparations were incubated with the reducing agent β-mercaptoethanol and boiled (β/b). α-Tubulin levels were used to control for equivalent loading ( N = 8 animals per group). f mRNA expression levels of Gpi1 in the cerebellum of Oxr1 +/+ or Oxr1 d/d mice by qRT-PCR ( N = 4 animals per group). g Gpi1 activity in N2a cells transfected with the vectors indicated compared to an empty vector control ( N = 3–5 independent repeats). h Gpi1 activity in N2a cells co-transfected with Gpi1 and either Oxr1-FL or Oxr1-C ( N = 3 independent repeats). Panels b , c , g , h : one-way ANOVA; Panels e , f: t-test; * p
    Figure Legend Snippet: Oxr1 modulates Gpi1 oligomerisation. a Dimerisation of Gpi1 in cells co-transfected with Gpi1 and either an empty vector or full-length (Oxr1-FL) or short (Oxr1-C) Oxr1 isoforms. Cells were treated with a cross-linker (DSP) and proteins were extracted in PBS; the loading buffer did not contain any reducing β-mercaptoethanol and samples were not boiled (non-reducing conditions). As a control, protein extracts from cells treated with DSP were incubated with the reducing agent β-mercaptoethanol and boiled (DSPβ/b). Vinculin (Vin) levels were used to control for equivalent loading. b – c Quantification of the dimeric ( b ) or tetrameric ( c ) versus monomeric forms of Gpi1 ( N = 6 independent repeats). d – e Western blot and quantification showing Gpi1 oligomerization in cerebellum from Oxr1 d/d and Oxr1 +/+ mice from proteins extracted in PBS and non-reducing conditions. Ponceau staining was used to control for equal loading. As a control, protein extracts from the same preparations were incubated with the reducing agent β-mercaptoethanol and boiled (β/b). α-Tubulin levels were used to control for equivalent loading ( N = 8 animals per group). f mRNA expression levels of Gpi1 in the cerebellum of Oxr1 +/+ or Oxr1 d/d mice by qRT-PCR ( N = 4 animals per group). g Gpi1 activity in N2a cells transfected with the vectors indicated compared to an empty vector control ( N = 3–5 independent repeats). h Gpi1 activity in N2a cells co-transfected with Gpi1 and either Oxr1-FL or Oxr1-C ( N = 3 independent repeats). Panels b , c , g , h : one-way ANOVA; Panels e , f: t-test; * p

    Techniques Used: Transfection, Plasmid Preparation, Incubation, Western Blot, Mouse Assay, Staining, Expressing, Quantitative RT-PCR, Activity Assay

    26) Product Images from "mTORC1 Is Essential for Early Steps during Schwann Cell Differentiation of Amniotic Fluid Stem Cells and Regulates Lipogenic Gene Expression"

    Article Title: mTORC1 Is Essential for Early Steps during Schwann Cell Differentiation of Amniotic Fluid Stem Cells and Regulates Lipogenic Gene Expression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0107004

    Scheme for the applied differentiation protocol. In order to initiate human AFS cell differentiation to a Schwann cell phenotype AFS cells were first treated in serum free α-MEM with 1 mM β-mercaptoethanol (Diff. I) for 24 hours. Afterwards cells were incubated in α-MEM supplemented with 10% fetal bovine serum and 35 ng/ml retinoic acid (Diff. II) for 72 hours. Subsequently, cells were cultured in α-MEM containing 10% fetal bovine serum supplemented with 20 ng/mL epidermal growth factor, 20 ng/mL basic fibroblast growth factor, 5 mM forskolin, 5 ng/mL platelet-derived growth factor-AA and 200 ng/mL recombinant human heregulin-beta1 (Diff. III) until day 15 of differentiation. Media was changed every 3 days, indicated by arrows. Pharmacologic (pharm.) treatment, consisting of rapamycin or statin, was applied together with Diff. III media.
    Figure Legend Snippet: Scheme for the applied differentiation protocol. In order to initiate human AFS cell differentiation to a Schwann cell phenotype AFS cells were first treated in serum free α-MEM with 1 mM β-mercaptoethanol (Diff. I) for 24 hours. Afterwards cells were incubated in α-MEM supplemented with 10% fetal bovine serum and 35 ng/ml retinoic acid (Diff. II) for 72 hours. Subsequently, cells were cultured in α-MEM containing 10% fetal bovine serum supplemented with 20 ng/mL epidermal growth factor, 20 ng/mL basic fibroblast growth factor, 5 mM forskolin, 5 ng/mL platelet-derived growth factor-AA and 200 ng/mL recombinant human heregulin-beta1 (Diff. III) until day 15 of differentiation. Media was changed every 3 days, indicated by arrows. Pharmacologic (pharm.) treatment, consisting of rapamycin or statin, was applied together with Diff. III media.

    Techniques Used: Cell Differentiation, Incubation, Cell Culture, Derivative Assay, Recombinant

    27) Product Images from "Myristoylation Restricts Orientation of the GRASP Domain on Membranes and Promotes Membrane Tethering *"

    Article Title: Myristoylation Restricts Orientation of the GRASP Domain on Membranes and Promotes Membrane Tethering *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.543561

    Complex structure of the GRASP55 GRASP domain with a membrane. A and B , fractional CVO profiles of stBLMs composed of DOPC and Ni 2+ -NTA-DGS (9:1) on HC18/β-mercaptoethanol (1:3) with 6.5 μmol/liter myrG55 ( A ) and 6 μmol/liter G55 ( B ). CVO profiles of the substrate (terminal gold film), oligo(ethylene oxide) tether, lipid bilayer components, and protein, indicated in the legend in A , are decomposed from multiple NR measurements. Protein CVO profiles determined from spline fits ( red lines ) and from atomistic models ( black dashed lines ) are shown in the same graphs for comparison. Lipid bilayer CVO profiles shown are those determined from the free-form fits and are very similar in the atomistic models. Dashed red lines indicate 68% confidence intervals for the free-form protein CVO profiles. C ), of myrG55 orientations (ϑ,ϕ) with respect to the bilayer normal. Only the half-sphere 0 ≤ ϑ ≤ 90° is shown. The best fit is observed at (ϑ,ϕ) = (26°,78°). The inset shows the definition of the Euler angles ϑ and ϕ. D , penetration depths of myrG55 into the bilayer at the orientations shown in panel C . The membrane surface is defined as the half-point in the decay of the lipid headgroup CVO toward bulk buffer ( brown traces in panels A and B at d ∼50 Å). E , orientation of myrG55 on the bilayer that corresponds to the best fit, (ϑ,ϕ) = (26°,78°), in the probability distribution of panel C . The long principal axis of the GRASP55 GRASP domain, ϑ 0 = 0°, is shown as a blue column . The internal ligand on the PDZ2 domain is shown in volume-filling representation , and the PDZ1 binding groove is shown as a green surface . The orientations of the PDZ2 ligand and a putative ligand bound to the PDZ1 groove, indicated as yellow cylinders , are roughly collinear. Approximate positions of the C- and N-terminal membrane binding sites, i.e. the Ni 2+ -ligated His tag and the myristoyl group, are shown in purple and maroon , respectively. F , probability distribution of G55 orientations (ϑ,ϕ) with respect to the bilayer normal on the half-sphere 0 ≤ ϑ ≤ 90°.
    Figure Legend Snippet: Complex structure of the GRASP55 GRASP domain with a membrane. A and B , fractional CVO profiles of stBLMs composed of DOPC and Ni 2+ -NTA-DGS (9:1) on HC18/β-mercaptoethanol (1:3) with 6.5 μmol/liter myrG55 ( A ) and 6 μmol/liter G55 ( B ). CVO profiles of the substrate (terminal gold film), oligo(ethylene oxide) tether, lipid bilayer components, and protein, indicated in the legend in A , are decomposed from multiple NR measurements. Protein CVO profiles determined from spline fits ( red lines ) and from atomistic models ( black dashed lines ) are shown in the same graphs for comparison. Lipid bilayer CVO profiles shown are those determined from the free-form fits and are very similar in the atomistic models. Dashed red lines indicate 68% confidence intervals for the free-form protein CVO profiles. C ), of myrG55 orientations (ϑ,ϕ) with respect to the bilayer normal. Only the half-sphere 0 ≤ ϑ ≤ 90° is shown. The best fit is observed at (ϑ,ϕ) = (26°,78°). The inset shows the definition of the Euler angles ϑ and ϕ. D , penetration depths of myrG55 into the bilayer at the orientations shown in panel C . The membrane surface is defined as the half-point in the decay of the lipid headgroup CVO toward bulk buffer ( brown traces in panels A and B at d ∼50 Å). E , orientation of myrG55 on the bilayer that corresponds to the best fit, (ϑ,ϕ) = (26°,78°), in the probability distribution of panel C . The long principal axis of the GRASP55 GRASP domain, ϑ 0 = 0°, is shown as a blue column . The internal ligand on the PDZ2 domain is shown in volume-filling representation , and the PDZ1 binding groove is shown as a green surface . The orientations of the PDZ2 ligand and a putative ligand bound to the PDZ1 groove, indicated as yellow cylinders , are roughly collinear. Approximate positions of the C- and N-terminal membrane binding sites, i.e. the Ni 2+ -ligated His tag and the myristoyl group, are shown in purple and maroon , respectively. F , probability distribution of G55 orientations (ϑ,ϕ) with respect to the bilayer normal on the half-sphere 0 ≤ ϑ ≤ 90°.

    Techniques Used: Binding Assay

    28) Product Images from "TDP-43 aggregation inside micronuclei reveals a potential mechanism for protein inclusion formation in ALS"

    Article Title: TDP-43 aggregation inside micronuclei reveals a potential mechanism for protein inclusion formation in ALS

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-56483-y

    TDP-43 co-localizes with f-LeuR and endogenous RGNEF within micronuclei and interacts in vitro and co-localizes in vivo with f-LeuR. ( A , B ) Representative confocal images of HEK293T cells showing co-localization (white arrows) of endogenous TDP-43 with f-LeuR (A) or endogenous RGNEF (B) within micronuclei after cellular metabolic stress using lactate. ( C ) IP of TDP-43-myc after crosslinking using DTSSP on protein lysate from HEK293T cells expressing f-LeuR and TDP-43-myc. WB was performed for detecting flag and then TDP-43 after stripping. Input controls are showed. β-mercaptoethanol was used to dissociate the crosslinked complex (* and ** mark electrophoretic shifts of approx. 440 kDa and 60 kDa, respectively). ( D ) Schematic of scAAV-9-LeuR virus and representative confocal images showing the extensive co-localization (white arrows) between LeuR and TDP-43 in brain of rats 4 weeks after the injection with the virus that express flag-LeuR in neurons under SYN1 promoter (yellow arrows indicate granular L-rich that doesn’t co-localize with TDP-43). The proteins were detected using goat anti-flag and rabbit anti-TDP-43 antibodies.
    Figure Legend Snippet: TDP-43 co-localizes with f-LeuR and endogenous RGNEF within micronuclei and interacts in vitro and co-localizes in vivo with f-LeuR. ( A , B ) Representative confocal images of HEK293T cells showing co-localization (white arrows) of endogenous TDP-43 with f-LeuR (A) or endogenous RGNEF (B) within micronuclei after cellular metabolic stress using lactate. ( C ) IP of TDP-43-myc after crosslinking using DTSSP on protein lysate from HEK293T cells expressing f-LeuR and TDP-43-myc. WB was performed for detecting flag and then TDP-43 after stripping. Input controls are showed. β-mercaptoethanol was used to dissociate the crosslinked complex (* and ** mark electrophoretic shifts of approx. 440 kDa and 60 kDa, respectively). ( D ) Schematic of scAAV-9-LeuR virus and representative confocal images showing the extensive co-localization (white arrows) between LeuR and TDP-43 in brain of rats 4 weeks after the injection with the virus that express flag-LeuR in neurons under SYN1 promoter (yellow arrows indicate granular L-rich that doesn’t co-localize with TDP-43). The proteins were detected using goat anti-flag and rabbit anti-TDP-43 antibodies.

    Techniques Used: In Vitro, In Vivo, Expressing, Western Blot, Stripping Membranes, Injection

    29) Product Images from "An Insight into the Molecular Basis of Salt Tolerance of l-myo-Inositol 1-P Synthase (PcINO1) from Porteresia coarctata (Roxb.) Tateoka, a Halophytic Wild Rice 1"

    Article Title: An Insight into the Molecular Basis of Salt Tolerance of l-myo-Inositol 1-P Synthase (PcINO1) from Porteresia coarctata (Roxb.) Tateoka, a Halophytic Wild Rice 1

    Journal:

    doi: 10.1104/pp.105.075150

    Surface hydrophobicity of PcINO1 mutant proteins. A, Bis-ANS binding titration of ΔPcINO1.2 and Os∷PcINO1 in presence and absence of added salt. A total of 0.15 mg/mL of each protein, in 20 m m Tris-HCl (pH 7.5), and 10 m m β -ME
    Figure Legend Snippet: Surface hydrophobicity of PcINO1 mutant proteins. A, Bis-ANS binding titration of ΔPcINO1.2 and Os∷PcINO1 in presence and absence of added salt. A total of 0.15 mg/mL of each protein, in 20 m m Tris-HCl (pH 7.5), and 10 m m β -ME

    Techniques Used: Mutagenesis, Binding Assay, Titration

    30) Product Images from "Anti-cancer mechanisms in two murine bone marrow derived-DC subsets activated with Toll-like receptor 4 agonists"

    Article Title: Anti-cancer mechanisms in two murine bone marrow derived-DC subsets activated with Toll-like receptor 4 agonists

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1701126

    Inhibition of 4T1-GFP cells growth by TLR4 activated BMDC is partially associated with IFN-β and partially peroxynitrite. (A) Dose-dependent inhibition of type-I-Interferon production (TBK1 kinase) by Bx795 during the coculture of 4T1-GFP with BMDC activated with IMM or LPS or non-activated. (*Note: asterisks indicate significant difference in comparison to respective 4T1-GFP growth suppression without Bx795). (B) Growth inhibition of 4T1-GFP cells by BMDC conditioned medium obtained after 24 h incubation with or without IMM or LPS in the presence or absence of Bx795. (C) Schematic diagram depicting inhibition steps in peroxynitrite pathway with indicated inhibitors. (D-F) Quantity of nitrites measured in the non-activated or TLR-4 activated BMDC conditioned media after 24 hours of incubation with different concentrations of L-NMMA (D) , FeTPPS (E) and β-Mercaptoethanol (F) . (G) Dose-dependent inhibition of NO-synthase with L-NMMA during the coculture of 4T1-GFP with BMDC activated with IMM or LPS or non-activated. (H) Dose-dependent destruction of peroxynitrites with FeTPPS during the coculture of 4T1-GFP with BMDC activated with IMM or LPS or non-activated. (I) Reduction of ROS by β-Mercaptoethanol during the coculture of 4T1-GFP with BMDC activated with IMM or LPS or non-activated (* Note: asterisks indicate significant difference in comparison to respective 4T1-GFP growth suppression without inhibitor). (J) Growth inhibition of 4T1-GFP cells by BMDC conditioned medium obtained after 24 h incubation with or without IMM or LPS in the presence or absence of indicated inhibitors (L-NMMA 100μM, FeTPPS 50μM).
    Figure Legend Snippet: Inhibition of 4T1-GFP cells growth by TLR4 activated BMDC is partially associated with IFN-β and partially peroxynitrite. (A) Dose-dependent inhibition of type-I-Interferon production (TBK1 kinase) by Bx795 during the coculture of 4T1-GFP with BMDC activated with IMM or LPS or non-activated. (*Note: asterisks indicate significant difference in comparison to respective 4T1-GFP growth suppression without Bx795). (B) Growth inhibition of 4T1-GFP cells by BMDC conditioned medium obtained after 24 h incubation with or without IMM or LPS in the presence or absence of Bx795. (C) Schematic diagram depicting inhibition steps in peroxynitrite pathway with indicated inhibitors. (D-F) Quantity of nitrites measured in the non-activated or TLR-4 activated BMDC conditioned media after 24 hours of incubation with different concentrations of L-NMMA (D) , FeTPPS (E) and β-Mercaptoethanol (F) . (G) Dose-dependent inhibition of NO-synthase with L-NMMA during the coculture of 4T1-GFP with BMDC activated with IMM or LPS or non-activated. (H) Dose-dependent destruction of peroxynitrites with FeTPPS during the coculture of 4T1-GFP with BMDC activated with IMM or LPS or non-activated. (I) Reduction of ROS by β-Mercaptoethanol during the coculture of 4T1-GFP with BMDC activated with IMM or LPS or non-activated (* Note: asterisks indicate significant difference in comparison to respective 4T1-GFP growth suppression without inhibitor). (J) Growth inhibition of 4T1-GFP cells by BMDC conditioned medium obtained after 24 h incubation with or without IMM or LPS in the presence or absence of indicated inhibitors (L-NMMA 100μM, FeTPPS 50μM).

    Techniques Used: Inhibition, Incubation

    31) Product Images from "Molecular Origin of the Self-Assembly of Lanreotide into Nanotubes: A Mutational Approach"

    Article Title: Molecular Origin of the Self-Assembly of Lanreotide into Nanotubes: A Mutational Approach

    Journal:

    doi: 10.1529/biophysj.107.108175

    FT-Raman ( a and c ) and ATR-FTIR ( b and d ) spectra. ( a ) FT-Raman spectra ( λ = 1064 nm) of naphthalene powder ( trace 1 ), naphthalene in THF ( trace 2 ), β -mercaptoethanol ( β -M) 1 M ( trace 3 ), Lan- β
    Figure Legend Snippet: FT-Raman ( a and c ) and ATR-FTIR ( b and d ) spectra. ( a ) FT-Raman spectra ( λ = 1064 nm) of naphthalene powder ( trace 1 ), naphthalene in THF ( trace 2 ), β -mercaptoethanol ( β -M) 1 M ( trace 3 ), Lan- β

    Techniques Used:

    32) Product Images from "Imaging chromatin nanostructure with binding-activated localization microscopy based on DNA structure fluctuations"

    Article Title: Imaging chromatin nanostructure with binding-activated localization microscopy based on DNA structure fluctuations

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw1301

    Influence of the chemical environment on YOYO-1 fluorescence signal intensity in nuclei of HeLa cells. Time course confocal microscopy of YOYO-1 was carried out after addition of 50% DMSO, 8% β-mercaptoethanol (BME) or 333 and 666 mM β-mercaptoethylamine (MEA) in PBS ( A ), 1 mM, 10 mM or 100 mM ascorbic acid (AA) in water ( B ) or H 2 O/HCl buffers with pH ranging from 8.54 to 3.09 ( C ). The first data-point corresponds to the fluorescence signal before addition of a buffer. Light-gray rectangle denotes data-points acquired after four and eight washing steps with either PBS (A) or H 2 O (B, C). Each curve represents measurements in > 100 cells, in five to six fields of view. Error bars correspond to a standard deviation. FL: average fluorescence intensity per cell (arbitrary units).
    Figure Legend Snippet: Influence of the chemical environment on YOYO-1 fluorescence signal intensity in nuclei of HeLa cells. Time course confocal microscopy of YOYO-1 was carried out after addition of 50% DMSO, 8% β-mercaptoethanol (BME) or 333 and 666 mM β-mercaptoethylamine (MEA) in PBS ( A ), 1 mM, 10 mM or 100 mM ascorbic acid (AA) in water ( B ) or H 2 O/HCl buffers with pH ranging from 8.54 to 3.09 ( C ). The first data-point corresponds to the fluorescence signal before addition of a buffer. Light-gray rectangle denotes data-points acquired after four and eight washing steps with either PBS (A) or H 2 O (B, C). Each curve represents measurements in > 100 cells, in five to six fields of view. Error bars correspond to a standard deviation. FL: average fluorescence intensity per cell (arbitrary units).

    Techniques Used: Fluorescence, Confocal Microscopy, Microelectrode Array, Standard Deviation

    33) Product Images from "Molecular Beacon-Metal Nanowire Interface: Effect of Probe Sequence and Surface Coverage on Sensor Performance"

    Article Title: Molecular Beacon-Metal Nanowire Interface: Effect of Probe Sequence and Surface Coverage on Sensor Performance

    Journal:

    doi: 10.1021/la703854x

    Effect of β-mercaptoethanol (BME) spacers on the fluorescence hybridization signal of HIV probes bound to Au/Ag nanowires. Ratios are mole ratios of BME to beacon. BF sample represents beacon-coated nanowires that were backfilled with BME. Error
    Figure Legend Snippet: Effect of β-mercaptoethanol (BME) spacers on the fluorescence hybridization signal of HIV probes bound to Au/Ag nanowires. Ratios are mole ratios of BME to beacon. BF sample represents beacon-coated nanowires that were backfilled with BME. Error

    Techniques Used: Fluorescence, Hybridization

    34) Product Images from "Electron transport chain of Saccharomyces cerevisiae mitochondria is inhibited by H2O2 at succinate-cytochrome c oxidoreductase level without lipid peroxidation involvement"

    Article Title: Electron transport chain of Saccharomyces cerevisiae mitochondria is inhibited by H2O2 at succinate-cytochrome c oxidoreductase level without lipid peroxidation involvement

    Journal:

    doi: 10.1080/10715760701635082

    Effect of β -mercaptoethanol on the impairment induced by H 2 O 2 on partial reactions of ETC. Mitochondria were incubated after H 2 O 2 treatment with indicated concentrations of β -mercaptoethanol during 30 min at 4°C on 50 m m KH 2 PO
    Figure Legend Snippet: Effect of β -mercaptoethanol on the impairment induced by H 2 O 2 on partial reactions of ETC. Mitochondria were incubated after H 2 O 2 treatment with indicated concentrations of β -mercaptoethanol during 30 min at 4°C on 50 m m KH 2 PO

    Techniques Used: Incubation

    35) Product Images from "The Motif of 76KRKCSK in Bm65 Is an Efficient Nuclear Localization Signal Involved in Production of Infectious Virions"

    Article Title: The Motif of 76KRKCSK in Bm65 Is an Efficient Nuclear Localization Signal Involved in Production of Infectious Virions

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2019.02739

    Western blotting analysis of Bm65-flag and Bm65(M1)-flag using antibodies against flag (Code#HT201 TransGen Biotech). (A) Strategy for expression of recombinant protein Bm65-flag and Bm65(M1)-flag. (B) Western blotting analysis of Bm65-flag expressed in BmNPV-infected BmN cells. (C) Western blotting analysis of Bm65-flag expressed in virus-infected BmN cells treated with β-mercaptoethanol. (D) Western blotting analysis of Bm65(M1)-flag expressed in virus-infected BmN cells. The prestained protein standards are on the left. The virus of vBm Bm65(M1)–Flag–GFP used is one in which all three cysteines were changed to alanine.
    Figure Legend Snippet: Western blotting analysis of Bm65-flag and Bm65(M1)-flag using antibodies against flag (Code#HT201 TransGen Biotech). (A) Strategy for expression of recombinant protein Bm65-flag and Bm65(M1)-flag. (B) Western blotting analysis of Bm65-flag expressed in BmNPV-infected BmN cells. (C) Western blotting analysis of Bm65-flag expressed in virus-infected BmN cells treated with β-mercaptoethanol. (D) Western blotting analysis of Bm65(M1)-flag expressed in virus-infected BmN cells. The prestained protein standards are on the left. The virus of vBm Bm65(M1)–Flag–GFP used is one in which all three cysteines were changed to alanine.

    Techniques Used: Western Blot, Expressing, Recombinant, Infection

    36) Product Images from "Bacterial protein translocation requires only one copy of the SecY complex in vivo"

    Article Title: Bacterial protein translocation requires only one copy of the SecY complex in vivo

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201205140

    Saturation of SecY channels with a post-translational translocation intermediate. (A) Scheme of a post-translational translocation intermediate generated with SecA and the substrate OmpA-GFP. The translocating chain contains the signal sequence of OmpA at the N terminus and the “superfolder” GFP at the C terminus. Its insertion into the SecY channel is monitored by disulfide bridge formation between cysteines in the substrate and SecY. (B) The insertion of OmpA-GFP, containing a cysteine at position 21 (21C), into SecY containing a cysteine at position 68 (68C) was tested by disulfide bridge formation after the addition of the oxidant CuPh 3 to intact E. coli cells. Where indicated, the substrate or SecY lacked a cysteine or the cysteines were blocked with N -ethylmaleimide (NEM) before addition of CuPh 3 . As a control, a substrate was used with a defective signal sequence (RR-21C). Where indicated, disulfide bridges were reduced by β-mercaptoethanol (β-ME). All samples were analyzed by SDS-PAGE followed by blotting with SecY or GFP antibodies. (C) OmpA-GFP was expressed from the arabinose (Ara)-inducible promoter in cells producing SecY at approximately endogenous level using a GUG translational start codon (plasmid pACYC-SecYEG). After addition of CuPh 3 , the lysate was analyzed by SDS-PAGE and blotting with SecY antibodies. Where indicated, rifampicin (Rif) was added for different time periods. Red arrows and black asterisks indicate cross-links between SecY and substrate or endogenous proteins, respectively. The right panel shows quantification of the cross-linking efficiency between SecY and translocation substrates, based on the decrease of noncross-linked SecY in experiments such as shown in the left panel and Fig. 2 B . Three different experiments were analyzed (mean and SD). (D) OmpA-GFP with either a wild-type (WT) or defective (RR) signal sequence was expressed under the arabinose (Ara) promoter in cells producing wild-type SecY or SecY lacking its plug domain (ΔP). Controls were performed with an empty vector (vec) and without Ara induction. Biotin-maleimide was added to the cells, and the modification of proteins was probed by SDS-PAGE and blotting with HRP-conjugated streptavidin. The samples were also probed with SecY, GFP, and trigger factor (TF; loading control) antibodies. Where indicated, cells were pretreated with rifampicin (Rif) before addition of biotin-maleimide. p30, a prominently modified cytosolic protein. The blue arrowheads indicate biotinylation of translocation-incompetent OmpA-GFP carrying a defective signal sequence.
    Figure Legend Snippet: Saturation of SecY channels with a post-translational translocation intermediate. (A) Scheme of a post-translational translocation intermediate generated with SecA and the substrate OmpA-GFP. The translocating chain contains the signal sequence of OmpA at the N terminus and the “superfolder” GFP at the C terminus. Its insertion into the SecY channel is monitored by disulfide bridge formation between cysteines in the substrate and SecY. (B) The insertion of OmpA-GFP, containing a cysteine at position 21 (21C), into SecY containing a cysteine at position 68 (68C) was tested by disulfide bridge formation after the addition of the oxidant CuPh 3 to intact E. coli cells. Where indicated, the substrate or SecY lacked a cysteine or the cysteines were blocked with N -ethylmaleimide (NEM) before addition of CuPh 3 . As a control, a substrate was used with a defective signal sequence (RR-21C). Where indicated, disulfide bridges were reduced by β-mercaptoethanol (β-ME). All samples were analyzed by SDS-PAGE followed by blotting with SecY or GFP antibodies. (C) OmpA-GFP was expressed from the arabinose (Ara)-inducible promoter in cells producing SecY at approximately endogenous level using a GUG translational start codon (plasmid pACYC-SecYEG). After addition of CuPh 3 , the lysate was analyzed by SDS-PAGE and blotting with SecY antibodies. Where indicated, rifampicin (Rif) was added for different time periods. Red arrows and black asterisks indicate cross-links between SecY and substrate or endogenous proteins, respectively. The right panel shows quantification of the cross-linking efficiency between SecY and translocation substrates, based on the decrease of noncross-linked SecY in experiments such as shown in the left panel and Fig. 2 B . Three different experiments were analyzed (mean and SD). (D) OmpA-GFP with either a wild-type (WT) or defective (RR) signal sequence was expressed under the arabinose (Ara) promoter in cells producing wild-type SecY or SecY lacking its plug domain (ΔP). Controls were performed with an empty vector (vec) and without Ara induction. Biotin-maleimide was added to the cells, and the modification of proteins was probed by SDS-PAGE and blotting with HRP-conjugated streptavidin. The samples were also probed with SecY, GFP, and trigger factor (TF; loading control) antibodies. Where indicated, cells were pretreated with rifampicin (Rif) before addition of biotin-maleimide. p30, a prominently modified cytosolic protein. The blue arrowheads indicate biotinylation of translocation-incompetent OmpA-GFP carrying a defective signal sequence.

    Techniques Used: Translocation Assay, Generated, Sequencing, SDS Page, Acetylene Reduction Assay, Plasmid Preparation, Modification

    SecY complexes interact in vivo through different surfaces. (A) View of Thermotoga maritima SecY complex from the periplasm. The N- and C-terminal halves of SecY are colored blue and red, respectively, SecG in green, and SecE in yellow. Balls in magenta indicate positions that were mutated in the E. coli protein to cysteines. Note that two cysteines are part of an inserted segment. (B) The accessibility of cysteine residues introduced into SecY was tested in intact E. coli by modification with biotin-PEG 2 -maleimide, followed by incubation with streptavidin (SA). The samples were analyzed by SDS-PAGE and immunoblotting with SecY antibodies (anti-SecY). The two bands correspond to one or two SecY molecules bound to tetrameric streptavidin. (C) The interaction of SecYs with the indicated cysteines was tested by addition of bismaleimide-PEG 3 (BM-PEG 3 ) to resuspended intact E. coli cells, pretreated with rifampicin for 30 min. The samples were analyzed by SDS-PAGE and immunoblotting for SecY. SecY 2 , cross-linked SecY dimers. (D) The front-to-front interaction of SecYs with the indicated cysteines was tested by spontaneous disulfide bridge formation in vivo. Where indicated, the samples were treated with β-mercaptoethanol (β-ME). (E) Scheme of a SecY complex containing SecE, SecG, and SecY in a single polypeptide chain. The gray dotted segments are added linkers. Residue L106 at the back of SecE (see A) is indicated. (F) The back-to-back interaction of single-chain SecY complexes with a cysteine at position 106 of SecE was tested by disulfide bridge formation after addition of the oxidant CuPh 3 to cell lysates. Controls were performed with protein lacking a cysteine and with β-ME addition after cross-linking.
    Figure Legend Snippet: SecY complexes interact in vivo through different surfaces. (A) View of Thermotoga maritima SecY complex from the periplasm. The N- and C-terminal halves of SecY are colored blue and red, respectively, SecG in green, and SecE in yellow. Balls in magenta indicate positions that were mutated in the E. coli protein to cysteines. Note that two cysteines are part of an inserted segment. (B) The accessibility of cysteine residues introduced into SecY was tested in intact E. coli by modification with biotin-PEG 2 -maleimide, followed by incubation with streptavidin (SA). The samples were analyzed by SDS-PAGE and immunoblotting with SecY antibodies (anti-SecY). The two bands correspond to one or two SecY molecules bound to tetrameric streptavidin. (C) The interaction of SecYs with the indicated cysteines was tested by addition of bismaleimide-PEG 3 (BM-PEG 3 ) to resuspended intact E. coli cells, pretreated with rifampicin for 30 min. The samples were analyzed by SDS-PAGE and immunoblotting for SecY. SecY 2 , cross-linked SecY dimers. (D) The front-to-front interaction of SecYs with the indicated cysteines was tested by spontaneous disulfide bridge formation in vivo. Where indicated, the samples were treated with β-mercaptoethanol (β-ME). (E) Scheme of a SecY complex containing SecE, SecG, and SecY in a single polypeptide chain. The gray dotted segments are added linkers. Residue L106 at the back of SecE (see A) is indicated. (F) The back-to-back interaction of single-chain SecY complexes with a cysteine at position 106 of SecE was tested by disulfide bridge formation after addition of the oxidant CuPh 3 to cell lysates. Controls were performed with protein lacking a cysteine and with β-ME addition after cross-linking.

    Techniques Used: In Vivo, Modification, Incubation, SDS Page

    37) Product Images from "Rapid Purification of a New P-I Class Metalloproteinase from Bothrops moojeni Venom with Antiplatelet Activity"

    Article Title: Rapid Purification of a New P-I Class Metalloproteinase from Bothrops moojeni Venom with Antiplatelet Activity

    Journal: BioMed Research International

    doi: 10.1155/2014/352420

    Proteolysis of bovine fibrinogen by BmooMP α -II. Line 1: negative control-fibrinogen incubated with enzyme for 0 minutes. (a) Proteolysis of bovine fibrinogen by BmooMP α -II time-dependent. Lanes 2–5: fibrinogen incubated with enzyme for 15, 30, 60 and 120 minutes, respectively. (b) Proteolysis of bovine fibrinogen by BmooMP α -II and effect of inhibitors. Lanes 2: positive control-fibrinogen incubated with enzyme for 60 minutes, 3–8: fibrinogen incubated with enzyme for 60 minutes after preincubation of BmooMP α -II with 5 mmol/L EDTA, 5 mmol/L β -mercaptoethanol, 5 mmol/L 1,10-phenanthroline, 5 mmol/L benzamidine, 5 mmol/L leupeptin, and 5 mmol/L aprotinin for 15 minutes, respectively. (c) Effect of the pH on the stability of BmooMP α -II. Lanes 2–8: fibrinogen incubated with enzyme for 60 minutes after preincubation of BmooMP α -II in pH 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0, respectively. (d) Effect of temperature on the stability of the BmooMP α -II. Lanes 2–8: fibrinogen incubated with enzyme for 60 minutes after preincubation of BmooMP α -II for 15 minutes at 30, 40, 50, 60, 70, 80, and 90°C, respectively.
    Figure Legend Snippet: Proteolysis of bovine fibrinogen by BmooMP α -II. Line 1: negative control-fibrinogen incubated with enzyme for 0 minutes. (a) Proteolysis of bovine fibrinogen by BmooMP α -II time-dependent. Lanes 2–5: fibrinogen incubated with enzyme for 15, 30, 60 and 120 minutes, respectively. (b) Proteolysis of bovine fibrinogen by BmooMP α -II and effect of inhibitors. Lanes 2: positive control-fibrinogen incubated with enzyme for 60 minutes, 3–8: fibrinogen incubated with enzyme for 60 minutes after preincubation of BmooMP α -II with 5 mmol/L EDTA, 5 mmol/L β -mercaptoethanol, 5 mmol/L 1,10-phenanthroline, 5 mmol/L benzamidine, 5 mmol/L leupeptin, and 5 mmol/L aprotinin for 15 minutes, respectively. (c) Effect of the pH on the stability of BmooMP α -II. Lanes 2–8: fibrinogen incubated with enzyme for 60 minutes after preincubation of BmooMP α -II in pH 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0, respectively. (d) Effect of temperature on the stability of the BmooMP α -II. Lanes 2–8: fibrinogen incubated with enzyme for 60 minutes after preincubation of BmooMP α -II for 15 minutes at 30, 40, 50, 60, 70, 80, and 90°C, respectively.

    Techniques Used: Negative Control, Incubation, Positive Control

    38) Product Images from "Specific and Covalent Targeting of Conjugating and Deconjugating Enzymes of Ubiquitin-Like Proteins"

    Article Title: Specific and Covalent Targeting of Conjugating and Deconjugating Enzymes of Ubiquitin-Like Proteins

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.24.1.84-95.2004

    UBL-VSs react specifically with cognate deconjugating enzymes. (A) Recombinant, purified UCH-L3 enzyme (left panel) and the catalytic domain of SENP2 (right panel) were incubated for 1 h at 37°C with SUMO-1 derivatives. No SUMO-1 probe was added to the first sample in each panel. Where indicated, enzymes were pretreated with 10 mM NEM prior to addition of SUMO-1-VS. SUMO 1-96 lacks the C-terminal glycine and VS moiety and therefore is not reactive. The reactions were terminated by addition of SDS sample buffer containing β-mercaptoethanol and boiling for 5 min. Polypeptides were resolved by SDS-11% PAGE and visualized by silver staining. The positions of molecular mass markers (in kilodaltons) are indicated on the right. The positions of UCH-L3 and the catalytic domain of SENP2 are indicated. The triangle marks the position of the SENP2-SUMO-1-VS adduct. (B) Same as panel A but with the Nedd8 derivatives Nedd8-VS and Nedd8 1-75 . Nedd8 1-75 lacks the C-terminal glycine and VS group. The triangle marks the position of the UCH-L3-Nedd8-VS adduct. (C) Same as panel A but with the Ub derivatives Ub-VS and Ub 1-75 . Ub 1-75 lacks the C-terminal glycine and VS group. The triangle marks the position of the UCH-L3-Ub-VS adduct.
    Figure Legend Snippet: UBL-VSs react specifically with cognate deconjugating enzymes. (A) Recombinant, purified UCH-L3 enzyme (left panel) and the catalytic domain of SENP2 (right panel) were incubated for 1 h at 37°C with SUMO-1 derivatives. No SUMO-1 probe was added to the first sample in each panel. Where indicated, enzymes were pretreated with 10 mM NEM prior to addition of SUMO-1-VS. SUMO 1-96 lacks the C-terminal glycine and VS moiety and therefore is not reactive. The reactions were terminated by addition of SDS sample buffer containing β-mercaptoethanol and boiling for 5 min. Polypeptides were resolved by SDS-11% PAGE and visualized by silver staining. The positions of molecular mass markers (in kilodaltons) are indicated on the right. The positions of UCH-L3 and the catalytic domain of SENP2 are indicated. The triangle marks the position of the SENP2-SUMO-1-VS adduct. (B) Same as panel A but with the Nedd8 derivatives Nedd8-VS and Nedd8 1-75 . Nedd8 1-75 lacks the C-terminal glycine and VS group. The triangle marks the position of the UCH-L3-Nedd8-VS adduct. (C) Same as panel A but with the Ub derivatives Ub-VS and Ub 1-75 . Ub 1-75 lacks the C-terminal glycine and VS group. The triangle marks the position of the UCH-L3-Ub-VS adduct.

    Techniques Used: Recombinant, Purification, Incubation, Polyacrylamide Gel Electrophoresis, Silver Staining

    Synthesis of UBLs with a C-terminal VS. (A) Reaction scheme for UBL-VS synthesis. Step 1, the processed form of a UBL, minus the C-terminal amino acid (−1aa), is expressed as a fusion protein with an intein and a CBD. Soluble fusion protein binds to a chitin affinity column. Step 2, spontaneous N-S acyl rearrangement resulting in an intermediate in which the peptide bond is replaced by a thioester linkage. Step 3, the UBL is released from the column by a transthioesterification reaction induced by incubation with MESNa sodium salt, resulting in the UBL-MESNa product. Step 4, the MESNa group is replaced by glycine-VS in a chemical ligation reaction, producing UBL-VS. Step 5, nucleophilic active-site residues of enzymes can covalently react with the VS group. (B) Nedd8-MESNa purification. FT, flowthrough after loading of lysate on chitin column; resin, chitin resin after Nedd8-MESNa elution. Eluted Nedd8-MESNa fractions were collected after overnight on-column cleavage induced by MESNa. Samples were prepared in SDS sample buffer without β-mercaptoethanol. (C) LC-ESI-MS analysis of Nedd8-MESNa and Nedd8-VS conversion product. The indicated multicharged species of Nedd8-MESNa correspond to a molecular weight of 8,627.6 ± 2.3, in agreement with a predicted molecular weight of 8,628.1. For Nedd8-VS, multicharged species correspond to a molecular weight of 8,620.8 ± 2.5, in agreement with a predicted molecular weight of 8,621.1.
    Figure Legend Snippet: Synthesis of UBLs with a C-terminal VS. (A) Reaction scheme for UBL-VS synthesis. Step 1, the processed form of a UBL, minus the C-terminal amino acid (−1aa), is expressed as a fusion protein with an intein and a CBD. Soluble fusion protein binds to a chitin affinity column. Step 2, spontaneous N-S acyl rearrangement resulting in an intermediate in which the peptide bond is replaced by a thioester linkage. Step 3, the UBL is released from the column by a transthioesterification reaction induced by incubation with MESNa sodium salt, resulting in the UBL-MESNa product. Step 4, the MESNa group is replaced by glycine-VS in a chemical ligation reaction, producing UBL-VS. Step 5, nucleophilic active-site residues of enzymes can covalently react with the VS group. (B) Nedd8-MESNa purification. FT, flowthrough after loading of lysate on chitin column; resin, chitin resin after Nedd8-MESNa elution. Eluted Nedd8-MESNa fractions were collected after overnight on-column cleavage induced by MESNa. Samples were prepared in SDS sample buffer without β-mercaptoethanol. (C) LC-ESI-MS analysis of Nedd8-MESNa and Nedd8-VS conversion product. The indicated multicharged species of Nedd8-MESNa correspond to a molecular weight of 8,627.6 ± 2.3, in agreement with a predicted molecular weight of 8,628.1. For Nedd8-VS, multicharged species correspond to a molecular weight of 8,620.8 ± 2.5, in agreement with a predicted molecular weight of 8,621.1.

    Techniques Used: Affinity Column, Incubation, Ligation, Purification, Mass Spectrometry, Molecular Weight

    SUMO-1 modifies SuPr-1 via its active-site cysteine. P19 cells were transiently transfected with a vector expressing HA-SuPr-1 or the catalytically inactive mutant HA-SuPr-1C466S, cells were harvested, and nuclear extracts were prepared. Nuclear extracts (25 μg) were incubated for 1 h at 37°C without (−) or with (+) SUMO-1-VS. The reactions were terminated by addition of SDS sample buffer containing β-mercaptoethanol and boiling for 5 min. Polypeptides were resolved by SDS-10% PAGE and immunoblotted (IB) with an anti-HA (α-HA) antibody.
    Figure Legend Snippet: SUMO-1 modifies SuPr-1 via its active-site cysteine. P19 cells were transiently transfected with a vector expressing HA-SuPr-1 or the catalytically inactive mutant HA-SuPr-1C466S, cells were harvested, and nuclear extracts were prepared. Nuclear extracts (25 μg) were incubated for 1 h at 37°C without (−) or with (+) SUMO-1-VS. The reactions were terminated by addition of SDS sample buffer containing β-mercaptoethanol and boiling for 5 min. Polypeptides were resolved by SDS-10% PAGE and immunoblotted (IB) with an anti-HA (α-HA) antibody.

    Techniques Used: Transfection, Plasmid Preparation, Expressing, Mutagenesis, Incubation, Polyacrylamide Gel Electrophoresis

    39) Product Images from "Investigating the mincing method for isolation of adipose-derived stem cells from pregnant women fat"

    Article Title: Investigating the mincing method for isolation of adipose-derived stem cells from pregnant women fat

    Journal: Cytotechnology

    doi: 10.1007/s10616-017-0162-8

    The morphology and RT-PCR analyses of the expression of insulin producing cells-related genes in P-ADSCs during the induced differentiation process. The step 1 decrease the FBS concentration from 10% to 2% and supplemented wtih NEAA and β-mercaptoethanol, then step 2 supplement with activin A, nicotinamide and bFGF, EGF, finally step 3 activin A, nicotinamide, and exendin 4 was supplied. a The changes in cell morphology could be observed from day 2 to day 16 after induction. b Expression analysis of islet markers using RT-PCR in pADSC after β-cell differentiation with indicated protocols. Scale bar 100 μm for micrograph
    Figure Legend Snippet: The morphology and RT-PCR analyses of the expression of insulin producing cells-related genes in P-ADSCs during the induced differentiation process. The step 1 decrease the FBS concentration from 10% to 2% and supplemented wtih NEAA and β-mercaptoethanol, then step 2 supplement with activin A, nicotinamide and bFGF, EGF, finally step 3 activin A, nicotinamide, and exendin 4 was supplied. a The changes in cell morphology could be observed from day 2 to day 16 after induction. b Expression analysis of islet markers using RT-PCR in pADSC after β-cell differentiation with indicated protocols. Scale bar 100 μm for micrograph

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Concentration Assay, Cell Differentiation

    40) Product Images from "The role of alanine 163 in solute permeability of Leishmania major aquaglyceroporin LmAQP1"

    Article Title: The role of alanine 163 in solute permeability of Leishmania major aquaglyceroporin LmAQP1

    Journal: Molecular and biochemical parasitology

    doi: 10.1016/j.molbiopara.2010.09.007

    Effect of Hg 2+ on water permeation of A163C and T164C LmAQP1. Swelling rates of Xenopus oocytes were determined in the absence of Hg 2+ , after 5 min incubation with 0.3 mM Hg 2+ , and 5 min incubation with 3 mM β-mercaptoethanol following Hg 2+ treatment.
    Figure Legend Snippet: Effect of Hg 2+ on water permeation of A163C and T164C LmAQP1. Swelling rates of Xenopus oocytes were determined in the absence of Hg 2+ , after 5 min incubation with 0.3 mM Hg 2+ , and 5 min incubation with 3 mM β-mercaptoethanol following Hg 2+ treatment.

    Techniques Used: Incubation

    Related Articles

    Electron Microscopy:

    Article Title: Dirofilaria immitis Microfilariae and Third-Stage Larvae Induce Canine NETosis Resulting in Different Types of Neutrophil Extracellular Traps
    Article Snippet: .. Scanning Electron Microscopy (SEM) Canine PMN (n = 3.5 × 105 ) were cocultured with vital D. immitis L3 (10 larvae/sample) or microfilariae (20 larvae/sample) on poly-l -lysine (Sigma-Aldrich) pre-coated coverslips (60 min, 37°C). .. After incubation, the samples were fixed in 2.5% glutaraldehyde [60 min, room temperature (RT), Merck], post-fixed in 1% osmium tetroxide (Merck), washed in distilled water, dehydrated, critical point dried by CO2 treatment, and spayed with gold.

    Whole Genome Amplification:

    Article Title: Genome wide identification of promoter binding sites for H4K12ac in human sperm and its relevance for early embryonic development
    Article Snippet: .. The required amount of DNA for microarray analysis (4 µg per sample) was generated using WGA Re-amplification Kit (Sigma-Aldrich, Steinheim, Germany). ..

    Buffer Exchange:

    Article Title: An accurate TMT-based approach to quantify and model lysine susceptibility to conjugation via N-hydroxysuccinimide esters in a monoclonal antibody
    Article Snippet: .. For the deglycosylation experiment, 400 μg of NIST mAb was diluted to 5 mg/mL through the addition of 45 μL of PBS (control glycosylated sample) or PNGaseF (Sigma, deglycosylated sample) and incubated at 37 °C for 5 h, prior to the initial buffer exchange step. .. To remove sample heterogeneity due to glycans and C-terminal lysine residues prior to intact mass analysis, a 20 μg aliquot of each TMT-labeled sample was treated with 4 μL PNGaseF overnight at 37 °C followed by a 2 h incubation with 4 μL of Carboxypeptidase B (Sigma, 0.05 mg/mL).

    Incubation:

    Article Title: An accurate TMT-based approach to quantify and model lysine susceptibility to conjugation via N-hydroxysuccinimide esters in a monoclonal antibody
    Article Snippet: .. For the deglycosylation experiment, 400 μg of NIST mAb was diluted to 5 mg/mL through the addition of 45 μL of PBS (control glycosylated sample) or PNGaseF (Sigma, deglycosylated sample) and incubated at 37 °C for 5 h, prior to the initial buffer exchange step. .. To remove sample heterogeneity due to glycans and C-terminal lysine residues prior to intact mass analysis, a 20 μg aliquot of each TMT-labeled sample was treated with 4 μL PNGaseF overnight at 37 °C followed by a 2 h incubation with 4 μL of Carboxypeptidase B (Sigma, 0.05 mg/mL).

    Purification:

    Article Title: Molecular Determinants of Scaffold-induced Linear Ubiquitinylation of B Cell Lymphoma/Leukemia 10 (Bcl10) during T Cell Receptor and Oncogenic Caspase Recruitment Domain-containing Protein 11 (CARD11) Signaling *
    Article Snippet: .. Purified mouse CD4+ T cells (7 × 107 /sample) or Jurkat T cells (1 × 108 /sample) were stimulated with or without 50 ng/ml PMA (Sigma) and 1 μ m iono (Sigma) for 30 min at 37 °C. ..

    Microarray:

    Article Title: Genome wide identification of promoter binding sites for H4K12ac in human sperm and its relevance for early embryonic development
    Article Snippet: .. The required amount of DNA for microarray analysis (4 µg per sample) was generated using WGA Re-amplification Kit (Sigma-Aldrich, Steinheim, Germany). ..

    Generated:

    Article Title: Genome wide identification of promoter binding sites for H4K12ac in human sperm and its relevance for early embryonic development
    Article Snippet: .. The required amount of DNA for microarray analysis (4 µg per sample) was generated using WGA Re-amplification Kit (Sigma-Aldrich, Steinheim, Germany). ..

    Western Blot:

    Article Title: Ku86 exists as both a full-length and a protease-sensitive natural variant in multiple myeloma cells
    Article Snippet: .. Western immunoblotting Cell lysates (20.0 μg of protein/sample) were first resolved on a 12.5% SDS-PAGE gel, transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore Corporation, Billerica, MA) and then blocked using Tris buffered saline Tween-20 (TBST) buffer containing 5% non-fat milk. .. Membranes were next hybridized overnight in the cold room using various murine monoclonal antibodies (mAb) – i.e. S10B1 anti-Ku86 N-terminus (amino acid (aa) residues 8–221; NeoMarkers, Fremont, CA); anti-heavy chain of the human major histocompatibility complex (MHC) mAb (clone 22.63.4, Accurate Chemical and Scientific Co., Westbuty, NY; a kind gift from P. Macary, from the National University of Singapore); and anti-actin (Santa Cruz Biotechnology, Santa Cruz, CA) mAbs; washed three times in ice cold TBS-T; and then incubated with horseradish peroxidase (hrp) conjugated anti-mouse IgG mAb (1:2,000; Santa Cruz) for 1 hr.

    SDS Page:

    Article Title: Ku86 exists as both a full-length and a protease-sensitive natural variant in multiple myeloma cells
    Article Snippet: .. Western immunoblotting Cell lysates (20.0 μg of protein/sample) were first resolved on a 12.5% SDS-PAGE gel, transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore Corporation, Billerica, MA) and then blocked using Tris buffered saline Tween-20 (TBST) buffer containing 5% non-fat milk. .. Membranes were next hybridized overnight in the cold room using various murine monoclonal antibodies (mAb) – i.e. S10B1 anti-Ku86 N-terminus (amino acid (aa) residues 8–221; NeoMarkers, Fremont, CA); anti-heavy chain of the human major histocompatibility complex (MHC) mAb (clone 22.63.4, Accurate Chemical and Scientific Co., Westbuty, NY; a kind gift from P. Macary, from the National University of Singapore); and anti-actin (Santa Cruz Biotechnology, Santa Cruz, CA) mAbs; washed three times in ice cold TBS-T; and then incubated with horseradish peroxidase (hrp) conjugated anti-mouse IgG mAb (1:2,000; Santa Cruz) for 1 hr.

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    • laemmli buffer  (Millipore)
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    Millipore laemmli buffer
    Immunoreactivity of antibody against rat SGLT2 (rSGLT2-Ab) in kidneys and small intestine of female rats. A : optimal conditions for Western blots with total cell membranes (TCM) from rat kidney cortex. For SDS-PAGE, isolated TCM were prepared in <t>Laemmli</t>
    Laemmli Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 92 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ksr medium  (Millipore)
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    LCD shows direct differentiation outcomes of <t>H9</t> cells seeded at different densities A-D , Undifferentiated H9 cells with localized high cell density were subjected to IF using anti-PAX6 (A and D) and anti-OCT4 (B and D) antibodies. E-L , H9 cells seeded at low density (8×10 3 cells/ cm 2 ) (E-H) and high density (1×10 4 cells/ cm 2 ) (I-L) were treated with <t>KSR</t> and N2 medium supplemented with noggin and SB431542 for 5 days. The cells were then subjected to the IF assay using anti-PAX6 antibody (green, E, H, I and L) and anti-OCT4 antibody (red, F, H, J and L) in the H9-derived cells. M, The areas of the signals in the OCT4, PAX6 and DAPI channels were calculated using Image J software. The ratios of the OCT4 and PAX6 area to DAPI area are shown (X±SD, n=8; *, P
    Ksr Medium, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    buffer a  (Millipore)
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    Kinetic analysis of editing of HNV by ThrRS at 37 °C and pH 8. All reactions were performed using HNV as the substrate. Panel (A), linear rate of AMP formation in the presence (open circles) and absence (filled circles) of tRNA Thr . Panel (B), enzyme-independent hydrolysis of HNV-AMP in Buffer A. Panels (C) and (D), rate of deacylation of HNV-[ 32 P] tRNA Thr in the presence and absence of wild type ThrRS, respectively.
    Buffer A, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Immunoreactivity of antibody against rat SGLT2 (rSGLT2-Ab) in kidneys and small intestine of female rats. A : optimal conditions for Western blots with total cell membranes (TCM) from rat kidney cortex. For SDS-PAGE, isolated TCM were prepared in Laemmli

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Expression of Na+-d-glucose cotransporter SGLT2 in rodents is kidney-specific and exhibits sex and species differences

    doi: 10.1152/ajpcell.00450.2011

    Figure Lengend Snippet: Immunoreactivity of antibody against rat SGLT2 (rSGLT2-Ab) in kidneys and small intestine of female rats. A : optimal conditions for Western blots with total cell membranes (TCM) from rat kidney cortex. For SDS-PAGE, isolated TCM were prepared in Laemmli

    Article Snippet: Denaturation of membrane samples in Laemmli buffer [±β-mercaptoethanol (β-ME)] and heating at different temperatures (37°C for 30 min, 65°C for 15 min, 95°C for 5 min), separation of the proteins through 10% SDS-PAGE minigels, as well as electrophoretic wet transfer to an Immobilon membrane (Millipore, Bedford, MA) were performed as described previously ( , ).

    Techniques: Western Blot, SDS Page, Isolation

    LCD shows direct differentiation outcomes of H9 cells seeded at different densities A-D , Undifferentiated H9 cells with localized high cell density were subjected to IF using anti-PAX6 (A and D) and anti-OCT4 (B and D) antibodies. E-L , H9 cells seeded at low density (8×10 3 cells/ cm 2 ) (E-H) and high density (1×10 4 cells/ cm 2 ) (I-L) were treated with KSR and N2 medium supplemented with noggin and SB431542 for 5 days. The cells were then subjected to the IF assay using anti-PAX6 antibody (green, E, H, I and L) and anti-OCT4 antibody (red, F, H, J and L) in the H9-derived cells. M, The areas of the signals in the OCT4, PAX6 and DAPI channels were calculated using Image J software. The ratios of the OCT4 and PAX6 area to DAPI area are shown (X±SD, n=8; *, P

    Journal: Biochemical and biophysical research communications

    Article Title: Synergistic contribution of SMAD signaling blockade and high localized cell density in the differentiation of neuroectoderm from H9 cells

    doi: 10.1016/j.bbrc.2014.08.137

    Figure Lengend Snippet: LCD shows direct differentiation outcomes of H9 cells seeded at different densities A-D , Undifferentiated H9 cells with localized high cell density were subjected to IF using anti-PAX6 (A and D) and anti-OCT4 (B and D) antibodies. E-L , H9 cells seeded at low density (8×10 3 cells/ cm 2 ) (E-H) and high density (1×10 4 cells/ cm 2 ) (I-L) were treated with KSR and N2 medium supplemented with noggin and SB431542 for 5 days. The cells were then subjected to the IF assay using anti-PAX6 antibody (green, E, H, I and L) and anti-OCT4 antibody (red, F, H, J and L) in the H9-derived cells. M, The areas of the signals in the OCT4, PAX6 and DAPI channels were calculated using Image J software. The ratios of the OCT4 and PAX6 area to DAPI area are shown (X±SD, n=8; *, P

    Article Snippet: Briefly, H9 cells were cultured on MEFs in KSR medium (DMEM/F12, 20 % KSR, 0.1 mM β-mercaptoethanol, 10 ng/ml of FGF-2) and disaggregated using accutase (Millipore, Billerica, MA, USA) for 20 min, washed with KSR medium and pre-plated on gelatin-coated 6-well plates for 1 h at 37 °C in the presence of the ROCK inhibitor (Y-27632) to remove MEFs.

    Techniques: Derivative Assay, Software

    Synergistic contribution of SMAD signaling blockers and localized high cell density in NE differentiation A-F, Five days after the cell-clump-based differentiation of NE in KSR and N2 medium with (D-F) or without (A-C) SMAD signaling blockers, H9-derived cells were subjected to the IF assay using anti-PAX6 antibody (green, A-D). The nuclei were stained using DAPI (blue, C and D). The micrographs were divided into 20 (5×4) squares as indicated (C and D). E-F , The number of total cells and PAX6-positive cells in each square was quantified using Image J software. The ratio of PAX6-positive cells to total cells in each square was determined. The squares with equivalent ratios were binned together. The ratios of PAX6-positive cells to total cells in each bin are shown (F, X±SD, **P

    Journal: Biochemical and biophysical research communications

    Article Title: Synergistic contribution of SMAD signaling blockade and high localized cell density in the differentiation of neuroectoderm from H9 cells

    doi: 10.1016/j.bbrc.2014.08.137

    Figure Lengend Snippet: Synergistic contribution of SMAD signaling blockers and localized high cell density in NE differentiation A-F, Five days after the cell-clump-based differentiation of NE in KSR and N2 medium with (D-F) or without (A-C) SMAD signaling blockers, H9-derived cells were subjected to the IF assay using anti-PAX6 antibody (green, A-D). The nuclei were stained using DAPI (blue, C and D). The micrographs were divided into 20 (5×4) squares as indicated (C and D). E-F , The number of total cells and PAX6-positive cells in each square was quantified using Image J software. The ratio of PAX6-positive cells to total cells in each square was determined. The squares with equivalent ratios were binned together. The ratios of PAX6-positive cells to total cells in each bin are shown (F, X±SD, **P

    Article Snippet: Briefly, H9 cells were cultured on MEFs in KSR medium (DMEM/F12, 20 % KSR, 0.1 mM β-mercaptoethanol, 10 ng/ml of FGF-2) and disaggregated using accutase (Millipore, Billerica, MA, USA) for 20 min, washed with KSR medium and pre-plated on gelatin-coated 6-well plates for 1 h at 37 °C in the presence of the ROCK inhibitor (Y-27632) to remove MEFs.

    Techniques: Derivative Assay, Staining, Software

    Kinetic analysis of editing of HNV by ThrRS at 37 °C and pH 8. All reactions were performed using HNV as the substrate. Panel (A), linear rate of AMP formation in the presence (open circles) and absence (filled circles) of tRNA Thr . Panel (B), enzyme-independent hydrolysis of HNV-AMP in Buffer A. Panels (C) and (D), rate of deacylation of HNV-[ 32 P] tRNA Thr in the presence and absence of wild type ThrRS, respectively.

    Journal: Biochemistry

    Article Title: Fidelity escape by the unnatural amino acid ?-hydroxynorvaline: an efficient substrate for Escherichia coli threonyl-tRNA synthetase with toxic effects on growth †

    doi: 10.1021/bi101360a

    Figure Lengend Snippet: Kinetic analysis of editing of HNV by ThrRS at 37 °C and pH 8. All reactions were performed using HNV as the substrate. Panel (A), linear rate of AMP formation in the presence (open circles) and absence (filled circles) of tRNA Thr . Panel (B), enzyme-independent hydrolysis of HNV-AMP in Buffer A. Panels (C) and (D), rate of deacylation of HNV-[ 32 P] tRNA Thr in the presence and absence of wild type ThrRS, respectively.

    Article Snippet: For both proteins, the final fractions were pooled, dialyzed against Buffer A (10 mM Tris pH 8.0, 100 mM KCl, 10 mM MgCl2 , 3 mM β-mercaptoethanol), and then concentrated under low speed centrifugation using Millipore concentrators, prior to storage in 50% glycerol at −20 °C.

    Techniques: