2-mercaptoethanol Search Results


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  • 99
    Thermo Fisher 2 mercaptoethanol
    DNA binding affinity of mtSSB and Escherichia coli SSB. Changes in fluorescence anisotropy of a fluorescein-conjugated 90-nt oligonucleotide substrate were measured in response to the step-wise addition of ( A ) mtSSB or ( B ) E. coli SSB proteins, as described in ‘Materials and Methods’ section. Binding buffer contained 30 mM HEPES-KOH (pH 7.5), 1 mM <t>2-mercaptoethanol,</t> 5 mM MgCl 2 , 0.01% NP-40, 50 mM NaCl, 20 nM oligonucleotide and the indicated amounts of proteins. Protein concentrations are expressed as tetramers. Error bars are standard deviations of triplicate determinations. ( C ) DNA binding cooperativity was estimated by replotting binding data for mtSSB (blue circles) and E. coli SSB (red squares) as a Hill plot.
    2 Mercaptoethanol, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 21282 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore 2 mercaptoethanol
    GSH, but not GSSG or other antioxidants, inhibits ceramide generation induced by H 2 O 2 in lung epithelial cells. A549 cells were preincubated for 1 h in medium supplemented with 1% serum in the presence or absence of 10 mM of GSH, GSSG, DTT, or <t>2-mercaptoethanol,</t> followed by incubations with 250 μM H 2 O 2 for an additional 30 min. The cells were rinsed with ice-cold PBS to terminate the treatments and harvested by trypsinization. GSH (A) and ceramide (B) levels were determined as described in Materials and Methods. Values represent mean ± SEM. *Mean of the group that was significantly different from the mean of the H 2 O 2 -treated group ( P
    2 Mercaptoethanol, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 22850 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Bio-Rad 2 mercaptoethanol
    (A) Western blotting under reducing condition with 2.5% <t>2-mercaptoethanol</t> of hACE2 expression in cell supernatants of transfected cells as well as cell lysates. (B) Western blotting of hACE2-IgG1 without reducing conditions.
    2 Mercaptoethanol, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 2975 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Bio-Rad β mercaptoethanol
    SDP disulfide bond formation is not essential for SDP activity and is independent of SdpAB. (A) β-Galactosidase activity of SdpC cysteine mutants in the presence and absence of SdpAB. All strains contain P sdpRI - lacZ ( pyrD ::P sdpRI -lacZ + ). The figures are labeled for their relevant sdp genotypes and are as follows: ABC + (TPM1476), C + (TPM1352), ABC C141A (TPM1505), C C141A (TPM1158), ABC C147A (TPM1507), C C147A (TPM1112), ABC C141A C147A (TPM1506), and C C141A C147A (TPM1207). The β-galactosidase activity assays were performed in triplicate as described in Materials and Methods. The averages and standard deviations are shown. (B) Toxic effect of SDP cysteine single and double mutants on SDP-sensitive (SdpI − ) cells (CDE433) and SDP-resistant (SdpI + ) cells (TPM758). The figures are labeled for their relevant sdp genotypes and are as follows: ABC + (TPM1476), C + (TPM1352), ABC C141A (TPM1505), C C141A (TPM1158), ABC C147A (TPM1507), C C147A (TPM1112), ABC C141A C147A (TPM1506), and C C141A C147A (TPM1207). (C) SdpC 33–203 disulfide bond formation in the presence and absence of SdpAB. Whole-cell cultures were prepared as described in Materials and Methods. Final samples were resuspended in 2× sample buffer with (+) or without (−) <t>β-mercaptoethanol.</t> The figures are labeled with their relevant sdp genotypes, as follows: ABC + (TPM1476) and C + (TPM1352).
    β Mercaptoethanol, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 93/100, based on 5867 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore β mercaptoethanol
    In vitro copper reductase assay with the Xenopus laevis H3-H4 tetramer. (A) Standard curve for Cu 1+ detection for copper reductase assay conditions used with X. laevis tetramers with increasing amounts of CuCl 2 -Tricine pH 7.5 at a ratio 1:10 in presence of 2 mM <t>β-Mercaptoethanol</t> as reducing agent (see Methods). Error bars indicate mean ± SD of three replicate measurements. (B) Same as (A) but for reactions with increasing amounts of CuCl 2 -Tricine pH 7.5 at a ratio 1:15 (see Methods). (C) Progress curve of copper reduction using 30 µM TCEP as the electron donor with 1 µM of X. laevis tetramer, equimolar monomeric X. laevis histone H3, and equimolar monomeric S. cerevisiae histone H2A. (D) Henri-Michaelis-Menten curves of initial velocities of reactions with the X. laevis tetramer. (E) Calculated enzymatic parameters of the tetramer from the curve in (D). (F) Same as (C) but with X. laevis tetramer, or equimolar RNase A or DTT. (G) Same as (A) but for reactions with increasing amounts of CuCl 2 -ADA pH 8 at a ratio 1:1 (see Methods). (H) Same as (A) but for reactions with increasing amounts of CuCl 2 -NTA pH 7.6 at a ratio 1:1 (see Methods). (I) Progress curves of copper reduction by 5 µM of the indicated X. laevis tetramers in presence of 200 µM TCEP and 0.5 mM CuCl 2 in 0.5 mM NTA pH 7.6.
    β Mercaptoethanol, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 28107 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    FUJIFILM 2 mercaptoethanol
    Determination of the impact of SS -derived fractions on IAV proteins. ( A –F ) DMSO control and the SS -derived fractions were added to solutions containing purified IAV; reactions proceeded at 25 °C for 48 h. Aqu: Aqueous extract. ( A ) The viral titer of each reaction was evaluated. Error bars indicate mean ± SD; n = 3 per group. ( B ) The images to the left and right are the results of CBB staining of SDS-samples without <t>2-Me</t> and with 2-Me, respectively. M: Marker. ( C ) The images to the left and right are the results of WB to detect IAV HA proteins. ( D ) The HA titer was evaluated. Error bars indicate mean ± SD; n = 3 per group. ( E ) The image is the result of WB to detect IAV NA protein. ( F ) The NA activity was evaluated. Error bars indicate mean of triplicate measurement of same sample ± SD. ( A,D ) Student’s t test was performed to analyze statistical difference; ** p
    2 Mercaptoethanol, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 94/100, based on 1002 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher m 2 mercaptoethanol
    Determination of the impact of SS -derived fractions on IAV proteins. ( A –F ) DMSO control and the SS -derived fractions were added to solutions containing purified IAV; reactions proceeded at 25 °C for 48 h. Aqu: Aqueous extract. ( A ) The viral titer of each reaction was evaluated. Error bars indicate mean ± SD; n = 3 per group. ( B ) The images to the left and right are the results of CBB staining of SDS-samples without <t>2-Me</t> and with 2-Me, respectively. M: Marker. ( C ) The images to the left and right are the results of WB to detect IAV HA proteins. ( D ) The HA titer was evaluated. Error bars indicate mean ± SD; n = 3 per group. ( E ) The image is the result of WB to detect IAV NA protein. ( F ) The NA activity was evaluated. Error bars indicate mean of triplicate measurement of same sample ± SD. ( A,D ) Student’s t test was performed to analyze statistical difference; ** p
    M 2 Mercaptoethanol, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 410 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher pierce 2 mercaptoethanol
    Determination of the impact of SS -derived fractions on IAV proteins. ( A –F ) DMSO control and the SS -derived fractions were added to solutions containing purified IAV; reactions proceeded at 25 °C for 48 h. Aqu: Aqueous extract. ( A ) The viral titer of each reaction was evaluated. Error bars indicate mean ± SD; n = 3 per group. ( B ) The images to the left and right are the results of CBB staining of SDS-samples without <t>2-Me</t> and with 2-Me, respectively. M: Marker. ( C ) The images to the left and right are the results of WB to detect IAV HA proteins. ( D ) The HA titer was evaluated. Error bars indicate mean ± SD; n = 3 per group. ( E ) The image is the result of WB to detect IAV NA protein. ( F ) The NA activity was evaluated. Error bars indicate mean of triplicate measurement of same sample ± SD. ( A,D ) Student’s t test was performed to analyze statistical difference; ** p
    Pierce 2 Mercaptoethanol, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Nacalai mercaptoethanol
    Determination of the impact of SS -derived fractions on IAV proteins. ( A –F ) DMSO control and the SS -derived fractions were added to solutions containing purified IAV; reactions proceeded at 25 °C for 48 h. Aqu: Aqueous extract. ( A ) The viral titer of each reaction was evaluated. Error bars indicate mean ± SD; n = 3 per group. ( B ) The images to the left and right are the results of CBB staining of SDS-samples without <t>2-Me</t> and with 2-Me, respectively. M: Marker. ( C ) The images to the left and right are the results of WB to detect IAV HA proteins. ( D ) The HA titer was evaluated. Error bars indicate mean ± SD; n = 3 per group. ( E ) The image is the result of WB to detect IAV NA protein. ( F ) The NA activity was evaluated. Error bars indicate mean of triplicate measurement of same sample ± SD. ( A,D ) Student’s t test was performed to analyze statistical difference; ** p
    Mercaptoethanol, supplied by Nacalai, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Merck KGaA β mercaptoethanol
    Determination of the impact of SS -derived fractions on IAV proteins. ( A –F ) DMSO control and the SS -derived fractions were added to solutions containing purified IAV; reactions proceeded at 25 °C for 48 h. Aqu: Aqueous extract. ( A ) The viral titer of each reaction was evaluated. Error bars indicate mean ± SD; n = 3 per group. ( B ) The images to the left and right are the results of CBB staining of SDS-samples without <t>2-Me</t> and with 2-Me, respectively. M: Marker. ( C ) The images to the left and right are the results of WB to detect IAV HA proteins. ( D ) The HA titer was evaluated. Error bars indicate mean ± SD; n = 3 per group. ( E ) The image is the result of WB to detect IAV NA protein. ( F ) The NA activity was evaluated. Error bars indicate mean of triplicate measurement of same sample ± SD. ( A,D ) Student’s t test was performed to analyze statistical difference; ** p
    β Mercaptoethanol, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 93/100, based on 420 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Merck KGaA mercaptoethanol
    Determination of the impact of SS -derived fractions on IAV proteins. ( A –F ) DMSO control and the SS -derived fractions were added to solutions containing purified IAV; reactions proceeded at 25 °C for 48 h. Aqu: Aqueous extract. ( A ) The viral titer of each reaction was evaluated. Error bars indicate mean ± SD; n = 3 per group. ( B ) The images to the left and right are the results of CBB staining of SDS-samples without <t>2-Me</t> and with 2-Me, respectively. M: Marker. ( C ) The images to the left and right are the results of WB to detect IAV HA proteins. ( D ) The HA titer was evaluated. Error bars indicate mean ± SD; n = 3 per group. ( E ) The image is the result of WB to detect IAV NA protein. ( F ) The NA activity was evaluated. Error bars indicate mean of triplicate measurement of same sample ± SD. ( A,D ) Student’s t test was performed to analyze statistical difference; ** p
    Mercaptoethanol, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 92/100, based on 88 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Merck & Co β mercaptoethanol
    Protein extraction from human hair using <t>β-mercaptoethanol</t> (sol-ME) and 1,4-dithiothreitol (sol-DTT) containing solutions. Protein was extracted at 50 °C from delipidized human hair. Protein concentration was measured by the Bradford assay. Error bars present standard errors.
    β Mercaptoethanol, supplied by Merck & Co, used in various techniques. Bioz Stars score: 93/100, based on 518 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Valiant β mercaptoethanol
    Protein extraction from human hair using <t>β-mercaptoethanol</t> (sol-ME) and 1,4-dithiothreitol (sol-DTT) containing solutions. Protein was extracted at 50 °C from delipidized human hair. Protein concentration was measured by the Bradford assay. Error bars present standard errors.
    β Mercaptoethanol, supplied by Valiant, used in various techniques. Bioz Stars score: 93/100, based on 447 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Fisher Scientific β mercaptoethanol
    Protein extraction from human hair using <t>β-mercaptoethanol</t> (sol-ME) and 1,4-dithiothreitol (sol-DTT) containing solutions. Protein was extracted at 50 °C from delipidized human hair. Protein concentration was measured by the Bradford assay. Error bars present standard errors.
    β Mercaptoethanol, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 92/100, based on 332 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Merck & Co 2 mercaptoethanol
    Crammer dimerization as a function of pH ( A ) SEC is used to monitor the dimerization of crammer as a function of pH. Blue Dextran 2000 as the internal standard is eluted at the void volume. Molecular mass standards: 1, chymotrypsinogen A (25 kDa) and 2, RNase A (13.7 kDa). ( B ) 13% (w/v) Tricine-SDS/PAGE. Lane 1 contains molecular size markers (M). Lanes labelled monomer and dimer are HPLC-purified proteins, and the proteins contained in last two lanes are as labelled in the Figure. β-ME, <t>2-mercaptoethanol.</t> ( C ) The results from SEC and Tricine-SDS/PAGE suggest that C72S at neutral pH exists in a monomeric form. ( D ) Crammer extracted from Drosophila heads is monomeric in the absence of 2-mercaptoethanol. The control lane labelled ‘crammer’ contains the recombinant protein, which exists as both monomer and dimer as visualized by the antibody against crammer. In ( B ) and ( D ) the molecular mass is given in kDa on the left-hand side. AU, arbitrary unit.
    2 Mercaptoethanol, supplied by Merck & Co, used in various techniques. Bioz Stars score: 93/100, based on 210 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    DNA binding affinity of mtSSB and Escherichia coli SSB. Changes in fluorescence anisotropy of a fluorescein-conjugated 90-nt oligonucleotide substrate were measured in response to the step-wise addition of ( A ) mtSSB or ( B ) E. coli SSB proteins, as described in ‘Materials and Methods’ section. Binding buffer contained 30 mM HEPES-KOH (pH 7.5), 1 mM 2-mercaptoethanol, 5 mM MgCl 2 , 0.01% NP-40, 50 mM NaCl, 20 nM oligonucleotide and the indicated amounts of proteins. Protein concentrations are expressed as tetramers. Error bars are standard deviations of triplicate determinations. ( C ) DNA binding cooperativity was estimated by replotting binding data for mtSSB (blue circles) and E. coli SSB (red squares) as a Hill plot.

    Journal: Nucleic Acids Research

    Article Title: Single-molecule DREEM imaging reveals DNA wrapping around human mitochondrial single-stranded DNA binding protein

    doi: 10.1093/nar/gky875

    Figure Lengend Snippet: DNA binding affinity of mtSSB and Escherichia coli SSB. Changes in fluorescence anisotropy of a fluorescein-conjugated 90-nt oligonucleotide substrate were measured in response to the step-wise addition of ( A ) mtSSB or ( B ) E. coli SSB proteins, as described in ‘Materials and Methods’ section. Binding buffer contained 30 mM HEPES-KOH (pH 7.5), 1 mM 2-mercaptoethanol, 5 mM MgCl 2 , 0.01% NP-40, 50 mM NaCl, 20 nM oligonucleotide and the indicated amounts of proteins. Protein concentrations are expressed as tetramers. Error bars are standard deviations of triplicate determinations. ( C ) DNA binding cooperativity was estimated by replotting binding data for mtSSB (blue circles) and E. coli SSB (red squares) as a Hill plot.

    Article Snippet: Samples (0.2 ml) containing 30 mM HEPES-KOH (pH 7.5), 1 mM 2-mercaptoethanol, 50 mM NaCl, 4× SYPRO Orange (Molecular Probes) and 5.0 μM mtSSB (expressed as tetramers) were heated from 16°C to 92°C.

    Techniques: Binding Assay, Fluorescence

    ER-retention of APP by addition of the di-lysine motif KKAA induces SDS stable homo-dimers in an oxidizing cellular surrounding. a Schematic illustration of the APP domain structure and the used dilysine retention constructs. APP isoform 695 (APP695) was fused to a C-terminal myc-tag, followed by either a KKAA ER retention motif, or a “leaky” KKFF motif allowing trafficking to the cis -Golgi compartment. The E1 domain, acidic domain, E2 domain, transmembrane domain (TM), and the intracellular domain are indicated. b CHO-K1 cells stably over expressing the respective APP695 wt or APP695 retention constructs were subjected to cell surface biotinylation. To inhibit endocytosis from the cell surface, AP180 D/N (lane four) was transiently co-expressed. At 24-h post-transfection, cell surface proteins were labeled with sulfo NHS-SS-biotin at 4°C, prior to lysis. Biotinylated samples were precipitated with NeutrAvidin agarose beads and analyzed by SDS-PAGE and Western blot (PD: NeutrAvidin; right panel ). As input controls, 20 μg of cell lysates were used (input; left panel ). APP full length, CTFs and AP180 D/N were probed with 9E10 antibody. Monoclonal α-Tubulin antibody served as a loading control to verify the absence of endomembrane contaminants in the biotinylation. For the input control, all samples were treated with SDS sample loading buffer, containing β-mercaptoethanol (BME) without heat denaturing prior to SDS gel electrophoresis. To elute surface proteins from NeutrAvidin beads, samples were denatured for 5 min at 95°C with BME and separated on 4–12% SDS gels. Molecular weight standards (kDa) are indicated on the left . c Cell lysates (20 μg each lane) of CHO-K1 cells stably overexpressing APP ER (KKAA), were incubated with SDS sample loading buffer (+ BME) and heat denatured for 10 min at the indicated temperatures (4–85°C). APP monomers and SDS stable dimers were detected, using the monoclonal anti myc antibody (9E10), shown on one representative Western blot. d APP dimers and monomers were quantified by densitometric analysis of Western blots, as the one shown in c . In each case, intensities at 4°C were set as 100%. Diagram shows relative intensity (%) of APP monomers and dimers, plotted against different temperatures. Data are ( n ≥ 3) ± SEM, one-way ANOVA

    Journal: Cellular and Molecular Life Sciences

    Article Title: APP dimer formation is initiated in the endoplasmic reticulum and differs between APP isoforms

    doi: 10.1007/s00018-011-0882-4

    Figure Lengend Snippet: ER-retention of APP by addition of the di-lysine motif KKAA induces SDS stable homo-dimers in an oxidizing cellular surrounding. a Schematic illustration of the APP domain structure and the used dilysine retention constructs. APP isoform 695 (APP695) was fused to a C-terminal myc-tag, followed by either a KKAA ER retention motif, or a “leaky” KKFF motif allowing trafficking to the cis -Golgi compartment. The E1 domain, acidic domain, E2 domain, transmembrane domain (TM), and the intracellular domain are indicated. b CHO-K1 cells stably over expressing the respective APP695 wt or APP695 retention constructs were subjected to cell surface biotinylation. To inhibit endocytosis from the cell surface, AP180 D/N (lane four) was transiently co-expressed. At 24-h post-transfection, cell surface proteins were labeled with sulfo NHS-SS-biotin at 4°C, prior to lysis. Biotinylated samples were precipitated with NeutrAvidin agarose beads and analyzed by SDS-PAGE and Western blot (PD: NeutrAvidin; right panel ). As input controls, 20 μg of cell lysates were used (input; left panel ). APP full length, CTFs and AP180 D/N were probed with 9E10 antibody. Monoclonal α-Tubulin antibody served as a loading control to verify the absence of endomembrane contaminants in the biotinylation. For the input control, all samples were treated with SDS sample loading buffer, containing β-mercaptoethanol (BME) without heat denaturing prior to SDS gel electrophoresis. To elute surface proteins from NeutrAvidin beads, samples were denatured for 5 min at 95°C with BME and separated on 4–12% SDS gels. Molecular weight standards (kDa) are indicated on the left . c Cell lysates (20 μg each lane) of CHO-K1 cells stably overexpressing APP ER (KKAA), were incubated with SDS sample loading buffer (+ BME) and heat denatured for 10 min at the indicated temperatures (4–85°C). APP monomers and SDS stable dimers were detected, using the monoclonal anti myc antibody (9E10), shown on one representative Western blot. d APP dimers and monomers were quantified by densitometric analysis of Western blots, as the one shown in c . In each case, intensities at 4°C were set as 100%. Diagram shows relative intensity (%) of APP monomers and dimers, plotted against different temperatures. Data are ( n ≥ 3) ± SEM, one-way ANOVA

    Article Snippet: Alternatively, samples were incubated with sample buffer without β-mercaptoethanol and heat denatured for 10 min. Proteins were electrophoresed on 4–12% NuPage (Novex®, Invitrogen) gradient gels and transferred onto nitrocellulose membranes (Millipore, Bedford, MA, USA).

    Techniques: Construct, Stable Transfection, Expressing, Transfection, Labeling, Lysis, SDS Page, Western Blot, SDS-Gel, Electrophoresis, Molecular Weight, Incubation

    APP wt homodimers are formed in the ER but also occur at the cell surface. a N2a cells were transiently co-transfected with NT HA APP695 CT Split GFP 1–10 and NT HA APP695 CT Split GFP 11. The cells were fixed with paraformaldehyde and immunostained with ER marker Grp78, followed by incubation with AlexaFlour-594 conjugated secondary antibody. The green fluorescence represents the APP dimer, induced by bimolecular fluorescence complementation (BiFC). Co-staining with Grp78 revealed colocalization of the APP dimer ( inset ) with the ER. b Single transfections of N2a cells with either NT HA APP695 CT Split GFP 1–10 or NT HA APP695 CT Split GFP 11. APP expression was verified with HA antibody, confirming a non-perturbed distribution ( right panel , red ). The green channel does not show any specific fluorescence. Scale bar is 10 μm. c N2a cells were transiently transfected with NT HA APP695 CT Split GFP 1–10, NT HA APP695 CT Split GFP 11 or GFP only. Cell lysates were analyzed via Western blotting for APP full length ( top part ), APP CTFs ( middle part ) and GFP, or GFP-containing CTFs ( bottom part ). Note that GFP antibody does not recognize GFP 11 alone. d CHO-K1 cells stably overexpressing APP 695 wt were pretreated for 1 h at 37°C in the presence of 10 μg/ml brefeldin A (BFA), or DMSO (vehicle) for 1 h to block protein secretion from the ER. Subsequently, cells were either directly lysed (BFA), or medium containing BFA was removed, and cells were incubated for additional 30 min at 37°C with 10 μM chlorpromazine (BFA/CPZ), to allow transport to the cell surface, but inhibit further endocytosis. Lysates were analyzed either under non-reducing (+β mercaptoethanol; −95°C), or reducing conditions (+β-mercaptoethanol; +95°C). Note that cells pretreated with BFA, followed by chlorpromazine exposure (BFA/CPZ), display two upper SDS-resistant bands migrating with a slight size shift, indicating different glycosylation patterns (dimer i/m). Note that strong denaturing conditions (+95°C) result in the disappearance of the upper migrating bands, suggesting disulfide bond formation. The separating line indicates different exposure times to visualize dimer formation in treated cells, whereas APP ER overexpressing cells served as size reference for dimers. e Cells were treated as described under d , and subjected to cell surface biotinylation to confirm drug treatments. APP ER is completely absent from the surface, whereas only minor quantities of mature APP reach the surface upon BFA treatment. Removal of BFA, and subsequent incubation with chlorpromazine (BFA/CPZ), strongly increases surface exposed mature APP, compared to vehicle control. Note that no dimers are recovered in biotinylation, because NeutrAvidin beads were boiled in BME containing sample buffer, to release biotin conjugated proteins

    Journal: Cellular and Molecular Life Sciences

    Article Title: APP dimer formation is initiated in the endoplasmic reticulum and differs between APP isoforms

    doi: 10.1007/s00018-011-0882-4

    Figure Lengend Snippet: APP wt homodimers are formed in the ER but also occur at the cell surface. a N2a cells were transiently co-transfected with NT HA APP695 CT Split GFP 1–10 and NT HA APP695 CT Split GFP 11. The cells were fixed with paraformaldehyde and immunostained with ER marker Grp78, followed by incubation with AlexaFlour-594 conjugated secondary antibody. The green fluorescence represents the APP dimer, induced by bimolecular fluorescence complementation (BiFC). Co-staining with Grp78 revealed colocalization of the APP dimer ( inset ) with the ER. b Single transfections of N2a cells with either NT HA APP695 CT Split GFP 1–10 or NT HA APP695 CT Split GFP 11. APP expression was verified with HA antibody, confirming a non-perturbed distribution ( right panel , red ). The green channel does not show any specific fluorescence. Scale bar is 10 μm. c N2a cells were transiently transfected with NT HA APP695 CT Split GFP 1–10, NT HA APP695 CT Split GFP 11 or GFP only. Cell lysates were analyzed via Western blotting for APP full length ( top part ), APP CTFs ( middle part ) and GFP, or GFP-containing CTFs ( bottom part ). Note that GFP antibody does not recognize GFP 11 alone. d CHO-K1 cells stably overexpressing APP 695 wt were pretreated for 1 h at 37°C in the presence of 10 μg/ml brefeldin A (BFA), or DMSO (vehicle) for 1 h to block protein secretion from the ER. Subsequently, cells were either directly lysed (BFA), or medium containing BFA was removed, and cells were incubated for additional 30 min at 37°C with 10 μM chlorpromazine (BFA/CPZ), to allow transport to the cell surface, but inhibit further endocytosis. Lysates were analyzed either under non-reducing (+β mercaptoethanol; −95°C), or reducing conditions (+β-mercaptoethanol; +95°C). Note that cells pretreated with BFA, followed by chlorpromazine exposure (BFA/CPZ), display two upper SDS-resistant bands migrating with a slight size shift, indicating different glycosylation patterns (dimer i/m). Note that strong denaturing conditions (+95°C) result in the disappearance of the upper migrating bands, suggesting disulfide bond formation. The separating line indicates different exposure times to visualize dimer formation in treated cells, whereas APP ER overexpressing cells served as size reference for dimers. e Cells were treated as described under d , and subjected to cell surface biotinylation to confirm drug treatments. APP ER is completely absent from the surface, whereas only minor quantities of mature APP reach the surface upon BFA treatment. Removal of BFA, and subsequent incubation with chlorpromazine (BFA/CPZ), strongly increases surface exposed mature APP, compared to vehicle control. Note that no dimers are recovered in biotinylation, because NeutrAvidin beads were boiled in BME containing sample buffer, to release biotin conjugated proteins

    Article Snippet: Alternatively, samples were incubated with sample buffer without β-mercaptoethanol and heat denatured for 10 min. Proteins were electrophoresed on 4–12% NuPage (Novex®, Invitrogen) gradient gels and transferred onto nitrocellulose membranes (Millipore, Bedford, MA, USA).

    Techniques: Transfection, Marker, Incubation, Fluorescence, Bimolecular Fluorescence Complementation Assay, Staining, Expressing, Western Blot, Stable Transfection, Blocking Assay

    THF toxicity in human cell lines deficient in protection against formaldehyde. a , CRISPR–Cas9-mediated disruptions of the  ADH5  locus in the human B cell line NALM-6. Exon 3 was targeted (in red) and the genetic changes (of one clone) in the sequence of both alleles are shown below.  b .  c , Cellular sensitivity of wild-type, Δ ADH5  and Δ FANCB  NALM-6 strains to THF and formaldehyde. Four Δ ADH5  clones were used.  d , Suppression of THF and formaldehyde toxicity in wild-type and Δ ADH5  HAP1 cells by the addition of 100 μ M β -ME. Data in  c  and  d  are from three independent experiments, each carried out in triplicate, and represent mean ± s.e.m.

    Journal: Nature

    Article Title: Mammals divert endogenous genotoxic formaldehyde into one-carbon metabolism

    doi: 10.1038/nature23481

    Figure Lengend Snippet: THF toxicity in human cell lines deficient in protection against formaldehyde. a , CRISPR–Cas9-mediated disruptions of the ADH5 locus in the human B cell line NALM-6. Exon 3 was targeted (in red) and the genetic changes (of one clone) in the sequence of both alleles are shown below. b . c , Cellular sensitivity of wild-type, Δ ADH5 and Δ FANCB NALM-6 strains to THF and formaldehyde. Four Δ ADH5 clones were used. d , Suppression of THF and formaldehyde toxicity in wild-type and Δ ADH5 HAP1 cells by the addition of 100 μ M β -ME. Data in c and d are from three independent experiments, each carried out in triplicate, and represent mean ± s.e.m.

    Article Snippet: DT40 cells were grown in RPMI medium (Gibco) supplemented with 3% chicken serum (Gibco), 7% fetal calf serum (Gibco), 50 μ M β -mercaptoethanol and penicillin/streptomycin (Gibco).

    Techniques: CRISPR, Sequencing, Clone Assay

    Cellular sensitivity of DT40 mutants to THF and formaldehyde. a , Wild-type, Δ ADH5 and Δ FANCC . FANCD2-Ub, monoubiquitinated FANCD2. b , Cellular sensitivity of DT40 mutants in Fanconi anaemia core-complex genes ( FANCB and FANCL ) to THF and formaldehyde. c , Cellular sensitivity of other Fanconi-deficient DT40 mutants to THF and formaldehyde. d , Cellular sensitivity to THF and formaldehyde of a panel of DT40 mutant cell lines defective in different DNA repair pathways: Fanconi anaemia pathway ( Δ FANCB ), translesion synthesis ( Δ REV3 ), nucleotide excision repair ( Δ XPA ), homologous recombination ( Δ XRCC2 ) and non-homologous end joining (Δ KU70 ). e , Top, scheme showing the reaction of formaldehyde with β -mercaptoethanol (β -ME). Bottom, suppression of THF and formaldehyde toxicity in wild-type, Δ ADH5 and Δ FANCC DT40 cells by the addition of 100 μ M β -ME. Data in b – e are from three independent experiments, each carried out in triplicate, and represent mean ± s.e.m.

    Journal: Nature

    Article Title: Mammals divert endogenous genotoxic formaldehyde into one-carbon metabolism

    doi: 10.1038/nature23481

    Figure Lengend Snippet: Cellular sensitivity of DT40 mutants to THF and formaldehyde. a , Wild-type, Δ ADH5 and Δ FANCC . FANCD2-Ub, monoubiquitinated FANCD2. b , Cellular sensitivity of DT40 mutants in Fanconi anaemia core-complex genes ( FANCB and FANCL ) to THF and formaldehyde. c , Cellular sensitivity of other Fanconi-deficient DT40 mutants to THF and formaldehyde. d , Cellular sensitivity to THF and formaldehyde of a panel of DT40 mutant cell lines defective in different DNA repair pathways: Fanconi anaemia pathway ( Δ FANCB ), translesion synthesis ( Δ REV3 ), nucleotide excision repair ( Δ XPA ), homologous recombination ( Δ XRCC2 ) and non-homologous end joining (Δ KU70 ). e , Top, scheme showing the reaction of formaldehyde with β -mercaptoethanol (β -ME). Bottom, suppression of THF and formaldehyde toxicity in wild-type, Δ ADH5 and Δ FANCC DT40 cells by the addition of 100 μ M β -ME. Data in b – e are from three independent experiments, each carried out in triplicate, and represent mean ± s.e.m.

    Article Snippet: DT40 cells were grown in RPMI medium (Gibco) supplemented with 3% chicken serum (Gibco), 7% fetal calf serum (Gibco), 50 μ M β -mercaptoethanol and penicillin/streptomycin (Gibco).

    Techniques: Mutagenesis, Translesion Synthesis, Homologous Recombination, Non-Homologous End Joining

    Oxidative degradation of folate derivatives liberates formaldehyde. a , In vitro activation of the FAP-1 probe by a concentration range of formaldehyde or THF. Formaldehyde and THF were incubated in PBS at 37 °C for 24 h before the addition of FAP-1 and the measurement of fluorescence. b , FAP-1 activation by formaldehyde in the absence or presence of ascorbate or β -mercaptoethanol (β -ME). c , Release of formaldehyde by THF (400 μ M) in PBS at different temperatures over time, measured using FAP-1. t 1/2 , estimated time required to release 50% of the total formaldehyde. Data in a – c are from two duplicate experiments. d , Top, detection of intracellular formaldehyde by FAP-1. Bottom, flow cytometry plot showing the fluorescence intensity of HAP1 cells. e , Activation of intracellular FAP-1 in HAP1 cells by formaldehyde or THF in the presence of a concentration range of ascorbate. f , Activation of FAP-1 in HAP1 cells exposed to folate derivatives sensitive or resistant to oxidative degradation. Data in e and f are from three duplicate experiments. A detailed description of the flow cytometry analysis in d – f . g , Measurement by gas chromatography–mass spectrometry (GC–MS) of the formaldehyde released by folate derivatives (200 μ M) after 24 h incubation in PBS at 37 °C. In the case of the oxidation-resistant derivatives, formaldehyde was not detected (n.d.) (limit of detection: 1.66 μ M). Data are from two independent experiments, each carried out in triplicate. h , Model for the release of up to two molecules of formaldehyde by 5,10-me-THF. All data represent mean ± s.e.m. Statistical significance was assessed using two-tailed Student’s t -tests. * P ≤ 0.05; NS, not significant.

    Journal: Nature

    Article Title: Mammals divert endogenous genotoxic formaldehyde into one-carbon metabolism

    doi: 10.1038/nature23481

    Figure Lengend Snippet: Oxidative degradation of folate derivatives liberates formaldehyde. a , In vitro activation of the FAP-1 probe by a concentration range of formaldehyde or THF. Formaldehyde and THF were incubated in PBS at 37 °C for 24 h before the addition of FAP-1 and the measurement of fluorescence. b , FAP-1 activation by formaldehyde in the absence or presence of ascorbate or β -mercaptoethanol (β -ME). c , Release of formaldehyde by THF (400 μ M) in PBS at different temperatures over time, measured using FAP-1. t 1/2 , estimated time required to release 50% of the total formaldehyde. Data in a – c are from two duplicate experiments. d , Top, detection of intracellular formaldehyde by FAP-1. Bottom, flow cytometry plot showing the fluorescence intensity of HAP1 cells. e , Activation of intracellular FAP-1 in HAP1 cells by formaldehyde or THF in the presence of a concentration range of ascorbate. f , Activation of FAP-1 in HAP1 cells exposed to folate derivatives sensitive or resistant to oxidative degradation. Data in e and f are from three duplicate experiments. A detailed description of the flow cytometry analysis in d – f . g , Measurement by gas chromatography–mass spectrometry (GC–MS) of the formaldehyde released by folate derivatives (200 μ M) after 24 h incubation in PBS at 37 °C. In the case of the oxidation-resistant derivatives, formaldehyde was not detected (n.d.) (limit of detection: 1.66 μ M). Data are from two independent experiments, each carried out in triplicate. h , Model for the release of up to two molecules of formaldehyde by 5,10-me-THF. All data represent mean ± s.e.m. Statistical significance was assessed using two-tailed Student’s t -tests. * P ≤ 0.05; NS, not significant.

    Article Snippet: DT40 cells were grown in RPMI medium (Gibco) supplemented with 3% chicken serum (Gibco), 7% fetal calf serum (Gibco), 50 μ M β -mercaptoethanol and penicillin/streptomycin (Gibco).

    Techniques: In Vitro, Activation Assay, Concentration Assay, Incubation, Fluorescence, Flow Cytometry, Cytometry, Gas Chromatography, Mass Spectrometry, Gas Chromatography-Mass Spectrometry, Two Tailed Test

    GSH, but not GSSG or other antioxidants, inhibits ceramide generation induced by H 2 O 2 in lung epithelial cells. A549 cells were preincubated for 1 h in medium supplemented with 1% serum in the presence or absence of 10 mM of GSH, GSSG, DTT, or 2-mercaptoethanol, followed by incubations with 250 μM H 2 O 2 for an additional 30 min. The cells were rinsed with ice-cold PBS to terminate the treatments and harvested by trypsinization. GSH (A) and ceramide (B) levels were determined as described in Materials and Methods. Values represent mean ± SEM. *Mean of the group that was significantly different from the mean of the H 2 O 2 -treated group ( P

    Journal: American journal of respiratory cell and molecular biology

    Article Title: Ceramide-Mediated Apoptosis in Lung Epithelial Cells Is Regulated by Glutathione

    doi: 10.1165/ajrcmb.25.6.4321

    Figure Lengend Snippet: GSH, but not GSSG or other antioxidants, inhibits ceramide generation induced by H 2 O 2 in lung epithelial cells. A549 cells were preincubated for 1 h in medium supplemented with 1% serum in the presence or absence of 10 mM of GSH, GSSG, DTT, or 2-mercaptoethanol, followed by incubations with 250 μM H 2 O 2 for an additional 30 min. The cells were rinsed with ice-cold PBS to terminate the treatments and harvested by trypsinization. GSH (A) and ceramide (B) levels were determined as described in Materials and Methods. Values represent mean ± SEM. *Mean of the group that was significantly different from the mean of the H 2 O 2 -treated group ( P

    Article Snippet: Bis-benzimide (Hoechst 33258), L-D-buthionine sulfoximine (BSO), aminotriazole (ATZ), GSH, GSSG, NAC, dithiothreitol (DTT), 2-mercaptoethanol, and all chemical reagents were from Sigma Chemical Co. (St. Louis, MO).

    Techniques:

    (A) Western blotting under reducing condition with 2.5% 2-mercaptoethanol of hACE2 expression in cell supernatants of transfected cells as well as cell lysates. (B) Western blotting of hACE2-IgG1 without reducing conditions.

    Journal: bioRxiv

    Article Title: Novel ACE2-IgG1 fusions with improved in vitro and in vivo activity against SARS-CoV2

    doi: 10.1101/2020.06.15.152157

    Figure Lengend Snippet: (A) Western blotting under reducing condition with 2.5% 2-mercaptoethanol of hACE2 expression in cell supernatants of transfected cells as well as cell lysates. (B) Western blotting of hACE2-IgG1 without reducing conditions.

    Article Snippet: Western blots were performed using 7.5% SDS-PAGE gels (Bio-Rad) under the non-reducing or reducing condition with 2.5% 2-mercaptoethanol and transferred to PVDF membranes.

    Techniques: Western Blot, Expressing, Transfection

    SDP disulfide bond formation is not essential for SDP activity and is independent of SdpAB. (A) β-Galactosidase activity of SdpC cysteine mutants in the presence and absence of SdpAB. All strains contain P sdpRI - lacZ ( pyrD ::P sdpRI -lacZ + ). The figures are labeled for their relevant sdp genotypes and are as follows: ABC + (TPM1476), C + (TPM1352), ABC C141A (TPM1505), C C141A (TPM1158), ABC C147A (TPM1507), C C147A (TPM1112), ABC C141A C147A (TPM1506), and C C141A C147A (TPM1207). The β-galactosidase activity assays were performed in triplicate as described in Materials and Methods. The averages and standard deviations are shown. (B) Toxic effect of SDP cysteine single and double mutants on SDP-sensitive (SdpI − ) cells (CDE433) and SDP-resistant (SdpI + ) cells (TPM758). The figures are labeled for their relevant sdp genotypes and are as follows: ABC + (TPM1476), C + (TPM1352), ABC C141A (TPM1505), C C141A (TPM1158), ABC C147A (TPM1507), C C147A (TPM1112), ABC C141A C147A (TPM1506), and C C141A C147A (TPM1207). (C) SdpC 33–203 disulfide bond formation in the presence and absence of SdpAB. Whole-cell cultures were prepared as described in Materials and Methods. Final samples were resuspended in 2× sample buffer with (+) or without (−) β-mercaptoethanol. The figures are labeled with their relevant sdp genotypes, as follows: ABC + (TPM1476) and C + (TPM1352).

    Journal: Journal of Bacteriology

    Article Title: Production of the Cannibalism Toxin SDP Is a Multistep Process That Requires SdpA and SdpB

    doi: 10.1128/JB.00407-13

    Figure Lengend Snippet: SDP disulfide bond formation is not essential for SDP activity and is independent of SdpAB. (A) β-Galactosidase activity of SdpC cysteine mutants in the presence and absence of SdpAB. All strains contain P sdpRI - lacZ ( pyrD ::P sdpRI -lacZ + ). The figures are labeled for their relevant sdp genotypes and are as follows: ABC + (TPM1476), C + (TPM1352), ABC C141A (TPM1505), C C141A (TPM1158), ABC C147A (TPM1507), C C147A (TPM1112), ABC C141A C147A (TPM1506), and C C141A C147A (TPM1207). The β-galactosidase activity assays were performed in triplicate as described in Materials and Methods. The averages and standard deviations are shown. (B) Toxic effect of SDP cysteine single and double mutants on SDP-sensitive (SdpI − ) cells (CDE433) and SDP-resistant (SdpI + ) cells (TPM758). The figures are labeled for their relevant sdp genotypes and are as follows: ABC + (TPM1476), C + (TPM1352), ABC C141A (TPM1505), C C141A (TPM1158), ABC C147A (TPM1507), C C147A (TPM1112), ABC C141A C147A (TPM1506), and C C141A C147A (TPM1207). (C) SdpC 33–203 disulfide bond formation in the presence and absence of SdpAB. Whole-cell cultures were prepared as described in Materials and Methods. Final samples were resuspended in 2× sample buffer with (+) or without (−) β-mercaptoethanol. The figures are labeled with their relevant sdp genotypes, as follows: ABC + (TPM1476) and C + (TPM1352).

    Article Snippet: The samples were vortexed and centrifuged at 13,000 × g for 10 min. Precipitated extracts containing protein were resuspended in 100 μl sample buffer (65.8 mM Tris-HCl [pH 6.8], 2% SDS, 26.3% [wt/vol] glycerol, 0.01% bromophenol blue, and 5% β-mercaptoethanol) with or without β-mercaptoethanol (Bio-Rad).

    Techniques: Activity Assay, Labeling

    Accumulation of sV23 in mutant and wild type stage 10 egg chambers. Late stage 10 egg chambers (5-25) were resuspended in Laemmli sample containing 5% beta mercaptoethanol and boiled to generate soluble protein extracts. Protein representing the number

    Journal: Developmental biology

    Article Title: Drosophila vitelline membrane assembly: A critical role for an evolutionarily conserved cysteine in the \u201cVM domain\u201d of sV23

    doi: 10.1016/j.ydbio.2010.08.037

    Figure Lengend Snippet: Accumulation of sV23 in mutant and wild type stage 10 egg chambers. Late stage 10 egg chambers (5-25) were resuspended in Laemmli sample containing 5% beta mercaptoethanol and boiled to generate soluble protein extracts. Protein representing the number

    Article Snippet: Proteins soluble in Laemmli sample containing 5% beta-mercaptoethanol were separated by SDS-PAGE and transferred to nitrocellulose using a Bio-Rad Mini-Protean system.

    Techniques: Mutagenesis

    In vitro copper reductase assay with the Xenopus laevis H3-H4 tetramer. (A) Standard curve for Cu 1+ detection for copper reductase assay conditions used with X. laevis tetramers with increasing amounts of CuCl 2 -Tricine pH 7.5 at a ratio 1:10 in presence of 2 mM β-Mercaptoethanol as reducing agent (see Methods). Error bars indicate mean ± SD of three replicate measurements. (B) Same as (A) but for reactions with increasing amounts of CuCl 2 -Tricine pH 7.5 at a ratio 1:15 (see Methods). (C) Progress curve of copper reduction using 30 µM TCEP as the electron donor with 1 µM of X. laevis tetramer, equimolar monomeric X. laevis histone H3, and equimolar monomeric S. cerevisiae histone H2A. (D) Henri-Michaelis-Menten curves of initial velocities of reactions with the X. laevis tetramer. (E) Calculated enzymatic parameters of the tetramer from the curve in (D). (F) Same as (C) but with X. laevis tetramer, or equimolar RNase A or DTT. (G) Same as (A) but for reactions with increasing amounts of CuCl 2 -ADA pH 8 at a ratio 1:1 (see Methods). (H) Same as (A) but for reactions with increasing amounts of CuCl 2 -NTA pH 7.6 at a ratio 1:1 (see Methods). (I) Progress curves of copper reduction by 5 µM of the indicated X. laevis tetramers in presence of 200 µM TCEP and 0.5 mM CuCl 2 in 0.5 mM NTA pH 7.6.

    Journal: bioRxiv

    Article Title: The Histone H3-H4 Tetramer is a Copper Reductase Enzyme

    doi: 10.1101/350652

    Figure Lengend Snippet: In vitro copper reductase assay with the Xenopus laevis H3-H4 tetramer. (A) Standard curve for Cu 1+ detection for copper reductase assay conditions used with X. laevis tetramers with increasing amounts of CuCl 2 -Tricine pH 7.5 at a ratio 1:10 in presence of 2 mM β-Mercaptoethanol as reducing agent (see Methods). Error bars indicate mean ± SD of three replicate measurements. (B) Same as (A) but for reactions with increasing amounts of CuCl 2 -Tricine pH 7.5 at a ratio 1:15 (see Methods). (C) Progress curve of copper reduction using 30 µM TCEP as the electron donor with 1 µM of X. laevis tetramer, equimolar monomeric X. laevis histone H3, and equimolar monomeric S. cerevisiae histone H2A. (D) Henri-Michaelis-Menten curves of initial velocities of reactions with the X. laevis tetramer. (E) Calculated enzymatic parameters of the tetramer from the curve in (D). (F) Same as (C) but with X. laevis tetramer, or equimolar RNase A or DTT. (G) Same as (A) but for reactions with increasing amounts of CuCl 2 -ADA pH 8 at a ratio 1:1 (see Methods). (H) Same as (A) but for reactions with increasing amounts of CuCl 2 -NTA pH 7.6 at a ratio 1:1 (see Methods). (I) Progress curves of copper reduction by 5 µM of the indicated X. laevis tetramers in presence of 200 µM TCEP and 0.5 mM CuCl 2 in 0.5 mM NTA pH 7.6.

    Article Snippet: Standard curves for each condition were produced using 2 mM β-Mercaptoethanol to ensure complete reduction of Cu2+ .

    Techniques: In Vitro, Reductase Assay

    Determination of the impact of SS -derived fractions on IAV proteins. ( A –F ) DMSO control and the SS -derived fractions were added to solutions containing purified IAV; reactions proceeded at 25 °C for 48 h. Aqu: Aqueous extract. ( A ) The viral titer of each reaction was evaluated. Error bars indicate mean ± SD; n = 3 per group. ( B ) The images to the left and right are the results of CBB staining of SDS-samples without 2-Me and with 2-Me, respectively. M: Marker. ( C ) The images to the left and right are the results of WB to detect IAV HA proteins. ( D ) The HA titer was evaluated. Error bars indicate mean ± SD; n = 3 per group. ( E ) The image is the result of WB to detect IAV NA protein. ( F ) The NA activity was evaluated. Error bars indicate mean of triplicate measurement of same sample ± SD. ( A,D ) Student’s t test was performed to analyze statistical difference; ** p

    Journal: Viruses

    Article Title: Saxifraga spinulosa-Derived Components Rapidly Inactivate Multiple Viruses Including SARS-CoV-2

    doi: 10.3390/v12070699

    Figure Lengend Snippet: Determination of the impact of SS -derived fractions on IAV proteins. ( A –F ) DMSO control and the SS -derived fractions were added to solutions containing purified IAV; reactions proceeded at 25 °C for 48 h. Aqu: Aqueous extract. ( A ) The viral titer of each reaction was evaluated. Error bars indicate mean ± SD; n = 3 per group. ( B ) The images to the left and right are the results of CBB staining of SDS-samples without 2-Me and with 2-Me, respectively. M: Marker. ( C ) The images to the left and right are the results of WB to detect IAV HA proteins. ( D ) The HA titer was evaluated. Error bars indicate mean ± SD; n = 3 per group. ( E ) The image is the result of WB to detect IAV NA protein. ( F ) The NA activity was evaluated. Error bars indicate mean of triplicate measurement of same sample ± SD. ( A,D ) Student’s t test was performed to analyze statistical difference; ** p

    Article Snippet: After the reaction time, these mixtures were mixed with the equal volume of 2× SDS sample buffer with and/or without 2-mercaptoethanol (2-Me; Wako Pure Chemical Industries, Ltd.).

    Techniques: Derivative Assay, Purification, Staining, Marker, Western Blot, Activity Assay

    Protein extraction from human hair using β-mercaptoethanol (sol-ME) and 1,4-dithiothreitol (sol-DTT) containing solutions. Protein was extracted at 50 °C from delipidized human hair. Protein concentration was measured by the Bradford assay. Error bars present standard errors.

    Journal: International Journal of Molecular Sciences

    Article Title: Keratin Biomembranes as a Model for Studying Onychomycosis

    doi: 10.3390/ijms21103512

    Figure Lengend Snippet: Protein extraction from human hair using β-mercaptoethanol (sol-ME) and 1,4-dithiothreitol (sol-DTT) containing solutions. Protein was extracted at 50 °C from delipidized human hair. Protein concentration was measured by the Bradford assay. Error bars present standard errors.

    Article Snippet: 1 g of delipidized hair was mixed with 20 mL of Shindai solution (sol-ME) [ ] containing 25 mM Tris.HCl, pH 8.5 (Merck, Kenilworth, NJ, USA), 2.6 M thiourea (Sigma-Aldrich, St. Louis, MO, USA), 5 M urea (Sigma-Aldrich, St. Louis, MO, USA) and 0.717 M β-mercaptoethanol (Merck, Kenilworth, NJ, USA) or of a modified Shindai solution (sol-DTT) containing 0.102 M 1,4-dithiothreitol (Merck, Kenilworth, NJ, USA) instead of β-mercaptoethanol.

    Techniques: Protein Extraction, Protein Concentration, Bradford Assay

    Crammer dimerization as a function of pH ( A ) SEC is used to monitor the dimerization of crammer as a function of pH. Blue Dextran 2000 as the internal standard is eluted at the void volume. Molecular mass standards: 1, chymotrypsinogen A (25 kDa) and 2, RNase A (13.7 kDa). ( B ) 13% (w/v) Tricine-SDS/PAGE. Lane 1 contains molecular size markers (M). Lanes labelled monomer and dimer are HPLC-purified proteins, and the proteins contained in last two lanes are as labelled in the Figure. β-ME, 2-mercaptoethanol. ( C ) The results from SEC and Tricine-SDS/PAGE suggest that C72S at neutral pH exists in a monomeric form. ( D ) Crammer extracted from Drosophila heads is monomeric in the absence of 2-mercaptoethanol. The control lane labelled ‘crammer’ contains the recombinant protein, which exists as both monomer and dimer as visualized by the antibody against crammer. In ( B ) and ( D ) the molecular mass is given in kDa on the left-hand side. AU, arbitrary unit.

    Journal: Biochemical Journal

    Article Title: A molten globule-to-ordered structure transition of Drosophila melanogaster crammer is required for its ability to inhibit cathepsin

    doi: 10.1042/BJ20111360

    Figure Lengend Snippet: Crammer dimerization as a function of pH ( A ) SEC is used to monitor the dimerization of crammer as a function of pH. Blue Dextran 2000 as the internal standard is eluted at the void volume. Molecular mass standards: 1, chymotrypsinogen A (25 kDa) and 2, RNase A (13.7 kDa). ( B ) 13% (w/v) Tricine-SDS/PAGE. Lane 1 contains molecular size markers (M). Lanes labelled monomer and dimer are HPLC-purified proteins, and the proteins contained in last two lanes are as labelled in the Figure. β-ME, 2-mercaptoethanol. ( C ) The results from SEC and Tricine-SDS/PAGE suggest that C72S at neutral pH exists in a monomeric form. ( D ) Crammer extracted from Drosophila heads is monomeric in the absence of 2-mercaptoethanol. The control lane labelled ‘crammer’ contains the recombinant protein, which exists as both monomer and dimer as visualized by the antibody against crammer. In ( B ) and ( D ) the molecular mass is given in kDa on the left-hand side. AU, arbitrary unit.

    Article Snippet: 2-Mercaptoethanol, chloramphenicol and sodium azide were supplied by Merck Research Laboratories.

    Techniques: Size-exclusion Chromatography, SDS Page, High Performance Liquid Chromatography, Purification, Recombinant