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    Structured Review

    Millipore β mercaptoethanol
    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% <t>β-mercaptoethanol</t> 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.
    β Mercaptoethanol, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 288 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) 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

    2) 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

    3) 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

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    Positive Control:

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    Synthesized:

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    Blocking Assay:

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    Incubation:

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    Article Title: Structural and Functional Characterization of Factor H Mutations Associated with Atypical Hemolytic Uremic Syndrome
    Article Snippet: Reduction of samples was achieved in the presence of 700 μM β-mercaptoethanol (Sigma), and incubation was achieved at 100°C for 5 min. Gels were stained with 1% Coomassie brilliant blue R-250 (Sigma) or with 12 mM silver nitrate reagent. .. Western blot analysis was performed on nitrocellulose membranes, by using anti–factor H monoclonal antibody 35H9, as described elsewhere (Sánchez-Corral et al. ).

    Article Title: Oxidation Resistance 1 Modulates Glycolytic Pathways in the Cerebellum via an Interaction with Glucose-6-Phosphate Isomerase
    Article Snippet: Paragraph title: Co-Immunoprecipitation, Protein Dimerization, and Western Blotting ... Immunoprecipitated proteins were washed three times in RIPA buffer before being boiled in NuPAGE loading buffer (Life Technologies) supplemented with β-mercaptoethanol (Sigma).

    Article Title: High-level production and purification in a functional state of an extrasynaptic gamma-aminobutyric acid type A receptor containing α4β3δ subunits
    Article Snippet: .. Western blotting and deglycosylation Purified protein was resolved on hand-casted 10% SDS-PAGE gels, loading approximately 10 μg of protein (as determined by Pierce BCA assay, Thermo Scientific, Rockford, IL) with 4x Laemmli buffer (Bio-Rad, Hercules, CA) supplemented with 10% β-mercaptoethanol (Sigma). .. Following transfer onto PVDF membranes (Immobilon-FL, Millipore, Billerica, MA) in 20% MeOH transfer buffer, membranes were washed with MeOH, air dried, re-wetted and blocked with Odyssey blocking buffer (PBS, Li-Cor, Lincoln, NE) for 1 hour at room temperature.

    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
    Article Snippet: GST-bound active Rac1 and Raf were washed three times in buffer and eluted from the GST beads using 2×SDS sample buffer containing 5% β-mercaptoethanol (Sigma) and heated for 5 min. .. Samples were separated using 4–20% SDS-PAGE and subjected to Western blotting using the monoclonal anti-Rac1 and Ras Ab supplied in the kit.

    Transfection:

    Article Title: Oxidation Resistance 1 Modulates Glycolytic Pathways in the Cerebellum via an Interaction with Glucose-6-Phosphate Isomerase
    Article Snippet: Immunoprecipitated proteins were washed three times in RIPA buffer before being boiled in NuPAGE loading buffer (Life Technologies) supplemented with β-mercaptoethanol (Sigma). .. To assess the level of oligomerisation of Gpi1, HeLa cells were co-transfected with Gpi1 and an empty vector or with Oxr1-FL or Oxr1-C. After 2-day transfection, cells were washed once with cold PBS and scraped from the dish surface in PBS supplemented with either 1-mM dithiobis(succinimidyl propionate) (DSP, Thermo Scientific) and lysed by passing through a 23-gauge needle and incubated at room temperature for 30 min.

    Article Title: Diametrically opposed effects of hypoxia and oxidative stress on two viral transactivators
    Article Snippet: Paragraph title: Cell Culture and Transfections ... Pharmacologic agents including menadione, paraquot dichloride, sodium selenite, beta-mercaptoethanol, glutathione, and N,N,N',N'-tetrakis (2 pyridylmethyl) ethylenediamine (TPEN) were purchased from Sigma (St. Louis, MO), and added 6 hours post-transfection, and cells were harvested 18-20 hours post-addition.

    Concentration Assay:

    Article Title: Oxidation Resistance 1 Modulates Glycolytic Pathways in the Cerebellum via an Interaction with Glucose-6-Phosphate Isomerase
    Article Snippet: Immunoprecipitated proteins were washed three times in RIPA buffer before being boiled in NuPAGE loading buffer (Life Technologies) supplemented with β-mercaptoethanol (Sigma). .. The reaction was then quenched by adding Tris pH 7.8 to reach 50-mM final concentration and incubated at room temperature for 15 min.

    Cell Culture:

    Article Title: Autophagy activator promotes neuronal differentiation of adult adipose-derived stromal cells ★
    Article Snippet: Initially, cells were not induced by culturing in pre-induction medium containing 1 mM β-mercaptoethanol (Sigma, St. Louis, MO, USA), 20% fetal calf serum and Dulbecco's modified Eagle's medium-high glucose, for 24 hours until reaching 70–80% confluency. .. The cells were then cultured in formal induction medium (containing 5 mM β-mercaptoethanol and DMEM), in both control and rapamycin groups.

    Article Title: Diametrically opposed effects of hypoxia and oxidative stress on two viral transactivators
    Article Snippet: Paragraph title: Cell Culture and Transfections ... Pharmacologic agents including menadione, paraquot dichloride, sodium selenite, beta-mercaptoethanol, glutathione, and N,N,N',N'-tetrakis (2 pyridylmethyl) ethylenediamine (TPEN) were purchased from Sigma (St. Louis, MO), and added 6 hours post-transfection, and cells were harvested 18-20 hours post-addition.

    Inhibition:

    Article Title: Investigation of amino acid specificity in the CydX small protein shows sequence plasticity at the functional level
    Article Snippet: Zone of growth inhibition assay Assays measuring the zone of growth inhibition of different strains to β-mercaptoethanol (Sigma-Aldrich) were conducted essentially as previously described [ ]. .. A sterile disc of Whatman filter paper was placed in the center of plate and 10 μl 14 M β-mercaptoethanol (Sigma Aldrich) was applied to the center of the disk.

    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
    Article Snippet: .. Zone of inhibition assays were conducted to test the sensitivity of the strains to β-mercaptoethanol, hydrogen peroxide, and hydroxylamine (Sigma-Aldrich). .. Before the zone of inhibition assay was conducted, the cultures were diluted in order to normalize all samples to an equivalent OD600 per ml.

    Article Title: Investigation of amino acid specificity in the CydX small protein shows sequence plasticity at the functional level
    Article Snippet: .. Assays measuring the zone of growth inhibition of different strains to β-mercaptoethanol (Sigma-Aldrich) were conducted essentially as previously described [ ]. ..

    Growth Inhibition Assay:

    Article Title: Investigation of amino acid specificity in the CydX small protein shows sequence plasticity at the functional level
    Article Snippet: Paragraph title: Zone of growth inhibition assay ... A sterile disc of Whatman filter paper was placed in the center of plate and 10 μl 14 M β-mercaptoethanol (Sigma Aldrich) was applied to the center of the disk.

    Injection:

    Article Title: Metabolic and thermal stimuli control K2P2.1 (TREK-1) through modular sensory and gating domains
    Article Snippet: Immunoblot analysis Oocytes were injected with 0.5 ng cRNA and lyzed after 48 h by repetitive pipetting in a cold buffer of 150 mM NaCl, 1.06 mM KH2 PO4 , 2.07 mM Na2 HPO4 , 1% Triton X100, pH7.4 supplemented with antiproteases. .. Lysates were treated for 30 min at 50°C with a sample buffer containing 2% SDS and 2% β-mercaptoethanol, and analysed by immunoblotting with the HA7 anti-HA antibodies (Sigma).

    Binding Assay:

    Article Title: Oxidation Resistance 1 Modulates Glycolytic Pathways in the Cerebellum via an Interaction with Glucose-6-Phosphate Isomerase
    Article Snippet: Co-Immunoprecipitation, Protein Dimerization, and Western Blotting To quantify the binding of Gpi1 and Oxr1 in cells, HeLa cells over-expressing Gpi1 and Oxr1 were washed once in cold PBS and lysed with cold standard RIPA buffer (50-mM Tris pH 7.5, 150-mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, all from Sigma) supplemented with protease and phosphatase inhibitor cocktail (Cell Signaling) by passing the cells through a 23-gauge needle ( > 10 strokes). .. Immunoprecipitated proteins were washed three times in RIPA buffer before being boiled in NuPAGE loading buffer (Life Technologies) supplemented with β-mercaptoethanol (Sigma).

    Article Title: Bacterial protein translocation requires only one copy of the SecY complex in vivo
    Article Snippet: After quenching with 20 mM β-mercaptoethanol for 20 min at 4°C, the cells were washed three times with 1 mL of the buffer and then resuspended in 200 µl of buffer containing 50 mM Tris-HCl, pH 7.5, 10 mM β-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 1.5 kU rLysozyme (EMD Millipore), 16 U/ml Benzonase nuclease (EMD Millipore), and 0.5% Triton X- 100. .. 25 µg streptavidin (Genscript) was added to a 40-µl aliquot of the reaction, and binding was allowed for 1 h at 4°C.

    In Vivo:

    Article Title: Bacterial protein translocation requires only one copy of the SecY complex in vivo
    Article Snippet: Paragraph title: In vivo biotinylation and streptavidin gel-shifting assays ... After quenching with 20 mM β-mercaptoethanol for 20 min at 4°C, the cells were washed three times with 1 mL of the buffer and then resuspended in 200 µl of buffer containing 50 mM Tris-HCl, pH 7.5, 10 mM β-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 1.5 kU rLysozyme (EMD Millipore), 16 U/ml Benzonase nuclease (EMD Millipore), and 0.5% Triton X- 100.

    Purification:

    Article Title: High-level production and purification in a functional state of an extrasynaptic gamma-aminobutyric acid type A receptor containing α4β3δ subunits
    Article Snippet: .. Western blotting and deglycosylation Purified protein was resolved on hand-casted 10% SDS-PAGE gels, loading approximately 10 μg of protein (as determined by Pierce BCA assay, Thermo Scientific, Rockford, IL) with 4x Laemmli buffer (Bio-Rad, Hercules, CA) supplemented with 10% β-mercaptoethanol (Sigma). .. Following transfer onto PVDF membranes (Immobilon-FL, Millipore, Billerica, MA) in 20% MeOH transfer buffer, membranes were washed with MeOH, air dried, re-wetted and blocked with Odyssey blocking buffer (PBS, Li-Cor, Lincoln, NE) for 1 hour at room temperature.

    FACS:

    Article Title: Diametrically opposed effects of hypoxia and oxidative stress on two viral transactivators
    Article Snippet: Pharmacologic agents including menadione, paraquot dichloride, sodium selenite, beta-mercaptoethanol, glutathione, and N,N,N',N'-tetrakis (2 pyridylmethyl) ethylenediamine (TPEN) were purchased from Sigma (St. Louis, MO), and added 6 hours post-transfection, and cells were harvested 18-20 hours post-addition. .. Transfections were normalized using the GFP expression plasmid, 2145 by FACS profiling a fraction of each transfection to determine the fraction of live-transfected cells (GFP-positive cells that did not stain with propidium iodide).

    Polyacrylamide Gel Electrophoresis:

    Article Title: Structural and Functional Characterization of Factor H Mutations Associated with Atypical Hemolytic Uremic Syndrome
    Article Snippet: Paragraph title: PAGE and Immunoblotting ... Reduction of samples was achieved in the presence of 700 μM β-mercaptoethanol (Sigma), and incubation was achieved at 100°C for 5 min. Gels were stained with 1% Coomassie brilliant blue R-250 (Sigma) or with 12 mM silver nitrate reagent.

    Lysis:

    Article Title: Ubiquitin-like modifier FAT10 attenuates RIG-I mediated antiviral signaling by segregating activated RIG-I from its signaling platform
    Article Snippet: .. After multiple 4 rounds of washing in 1 ml of IP lysis buffer, proteins were eluted by boiling at 95 °C in laemmli buffer (Biophoretic) containing 5% β-mercaptoethanol (Sigma-Aldrich). ..

    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
    Article Snippet: For the Rac assays, empty vector (EV), RasGRF2, RasGRF2+p35+Cdk5, and RasGRF2+Cdk5 were cotransfected into CHO cells, and cells were harvested in the provided lysis–binding–wash buffer. .. GST-bound active Rac1 and Raf were washed three times in buffer and eluted from the GST beads using 2×SDS sample buffer containing 5% β-mercaptoethanol (Sigma) and heated for 5 min.

    SDS Page:

    Article Title: Bacterial protein translocation requires only one copy of the SecY complex in vivo
    Article Snippet: After quenching with 20 mM β-mercaptoethanol for 20 min at 4°C, the cells were washed three times with 1 mL of the buffer and then resuspended in 200 µl of buffer containing 50 mM Tris-HCl, pH 7.5, 10 mM β-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 1.5 kU rLysozyme (EMD Millipore), 16 U/ml Benzonase nuclease (EMD Millipore), and 0.5% Triton X- 100. .. The samples were then supplemented with 0.1% SDS, 12.5% glycerol, and bromophenol blue for SDS-PAGE.

    Article Title: High-level production and purification in a functional state of an extrasynaptic gamma-aminobutyric acid type A receptor containing α4β3δ subunits
    Article Snippet: .. Western blotting and deglycosylation Purified protein was resolved on hand-casted 10% SDS-PAGE gels, loading approximately 10 μg of protein (as determined by Pierce BCA assay, Thermo Scientific, Rockford, IL) with 4x Laemmli buffer (Bio-Rad, Hercules, CA) supplemented with 10% β-mercaptoethanol (Sigma). .. Following transfer onto PVDF membranes (Immobilon-FL, Millipore, Billerica, MA) in 20% MeOH transfer buffer, membranes were washed with MeOH, air dried, re-wetted and blocked with Odyssey blocking buffer (PBS, Li-Cor, Lincoln, NE) for 1 hour at room temperature.

    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
    Article Snippet: GST-bound active Rac1 and Raf were washed three times in buffer and eluted from the GST beads using 2×SDS sample buffer containing 5% β-mercaptoethanol (Sigma) and heated for 5 min. .. Samples were separated using 4–20% SDS-PAGE and subjected to Western blotting using the monoclonal anti-Rac1 and Ras Ab supplied in the kit.

    Plasmid Preparation:

    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
    Article Snippet: Zone of inhibition assays were conducted to test the sensitivity of the strains to β-mercaptoethanol, hydrogen peroxide, and hydroxylamine (Sigma-Aldrich). .. Assays using strains that did not contain a plasmid were plated on LB, while those strains containing a plasmid were plated on LB-ampicillin plates supplemented with arabinose (0.2%) (LB-ampicillin-arabinose) to induce protein expression from the pBAD24 plasmid.

    Article Title: Oxidation Resistance 1 Modulates Glycolytic Pathways in the Cerebellum via an Interaction with Glucose-6-Phosphate Isomerase
    Article Snippet: Immunoprecipitated proteins were washed three times in RIPA buffer before being boiled in NuPAGE loading buffer (Life Technologies) supplemented with β-mercaptoethanol (Sigma). .. To assess the level of oligomerisation of Gpi1, HeLa cells were co-transfected with Gpi1 and an empty vector or with Oxr1-FL or Oxr1-C. After 2-day transfection, cells were washed once with cold PBS and scraped from the dish surface in PBS supplemented with either 1-mM dithiobis(succinimidyl propionate) (DSP, Thermo Scientific) and lysed by passing through a 23-gauge needle and incubated at room temperature for 30 min.

    Article Title: Diametrically opposed effects of hypoxia and oxidative stress on two viral transactivators
    Article Snippet: Pharmacologic agents including menadione, paraquot dichloride, sodium selenite, beta-mercaptoethanol, glutathione, and N,N,N',N'-tetrakis (2 pyridylmethyl) ethylenediamine (TPEN) were purchased from Sigma (St. Louis, MO), and added 6 hours post-transfection, and cells were harvested 18-20 hours post-addition. .. Transfections were normalized using the GFP expression plasmid, 2145 by FACS profiling a fraction of each transfection to determine the fraction of live-transfected cells (GFP-positive cells that did not stain with propidium iodide).

    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
    Article Snippet: For the Rac assays, empty vector (EV), RasGRF2, RasGRF2+p35+Cdk5, and RasGRF2+Cdk5 were cotransfected into CHO cells, and cells were harvested in the provided lysis–binding–wash buffer. .. GST-bound active Rac1 and Raf were washed three times in buffer and eluted from the GST beads using 2×SDS sample buffer containing 5% β-mercaptoethanol (Sigma) and heated for 5 min.

    In Vitro:

    Article Title: Autophagy activator promotes neuronal differentiation of adult adipose-derived stromal cells ★
    Article Snippet: Paragraph title: In vitro induction and differentiation of adult adipose-derived stromal cells into neuronal-like cells, and rapamycin treatment ... Initially, cells were not induced by culturing in pre-induction medium containing 1 mM β-mercaptoethanol (Sigma, St. Louis, MO, USA), 20% fetal calf serum and Dulbecco's modified Eagle's medium-high glucose, for 24 hours until reaching 70–80% confluency.

    Immunoprecipitation:

    Article Title: Ubiquitin-like modifier FAT10 attenuates RIG-I mediated antiviral signaling by segregating activated RIG-I from its signaling platform
    Article Snippet: Paragraph title: Immunoprecipitation ... After multiple 4 rounds of washing in 1 ml of IP lysis buffer, proteins were eluted by boiling at 95 °C in laemmli buffer (Biophoretic) containing 5% β-mercaptoethanol (Sigma-Aldrich).

    Article Title: Oxidation Resistance 1 Modulates Glycolytic Pathways in the Cerebellum via an Interaction with Glucose-6-Phosphate Isomerase
    Article Snippet: .. Immunoprecipitated proteins were washed three times in RIPA buffer before being boiled in NuPAGE loading buffer (Life Technologies) supplemented with β-mercaptoethanol (Sigma). .. Proteins were run on pre-cast NuPAGE Bis-Tris gels (Life Technologies) and transferred as per the manufacturer’s protocol.

    Staining:

    Article Title: Structural and Functional Characterization of Factor H Mutations Associated with Atypical Hemolytic Uremic Syndrome
    Article Snippet: .. Reduction of samples was achieved in the presence of 700 μM β-mercaptoethanol (Sigma), and incubation was achieved at 100°C for 5 min. Gels were stained with 1% Coomassie brilliant blue R-250 (Sigma) or with 12 mM silver nitrate reagent. .. Western blot analysis was performed on nitrocellulose membranes, by using anti–factor H monoclonal antibody 35H9, as described elsewhere (Sánchez-Corral et al. ).

    Article Title: Diametrically opposed effects of hypoxia and oxidative stress on two viral transactivators
    Article Snippet: Pharmacologic agents including menadione, paraquot dichloride, sodium selenite, beta-mercaptoethanol, glutathione, and N,N,N',N'-tetrakis (2 pyridylmethyl) ethylenediamine (TPEN) were purchased from Sigma (St. Louis, MO), and added 6 hours post-transfection, and cells were harvested 18-20 hours post-addition. .. Transfections were normalized using the GFP expression plasmid, 2145 by FACS profiling a fraction of each transfection to determine the fraction of live-transfected cells (GFP-positive cells that did not stain with propidium iodide).

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  • 95
    Millipore buffer a
    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: 95/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/buffer a/product/Millipore
    Average 95 stars, based on 30 article reviews
    Price from $9.99 to $1999.99
    buffer a - by Bioz Stars, 2020-02
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    99
    Millipore ksr medium
    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: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ksr medium/product/Millipore
    Average 99 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    ksr medium - by Bioz Stars, 2020-02
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    99
    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
    https://www.bioz.com/result/laemmli buffer/product/Millipore
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    laemmli buffer - by Bioz Stars, 2020-02
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    Image Search Results


    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:

    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

    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