tris-glycine gel Search Results


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  • 99
    Thermo Fisher tris glycine gels
    Cysteine desulfhydrase activity by in situ staining. <t>MGL</t> activity was monitored in <t>Tris-glycine</t> gels under nondenaturing conditions using l -cysteine as the substrate. Lanes: M, molecular mass markers; 1, purified recombinant His-tagged MGL protein; 2,
    Tris Glycine Gels, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 7283 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore tris glycine gel
    Expression in cultured mammalian cells of artificial RNA-binding proteins containing PrLDs. ( a ) Schematic diagram showing artificial RNA-binding proteins containing PrLDs, as well as the deletion constructs. All constructs contained a GFP tag at the C-terminus. sPFD: synthetic prion-forming domain; cPFD: control prion-forming domain [exhibiting low degrees of prion propensity 17 ]. ( b ) Immunoblot analysis of SYG/SYGQ-NES expression in transfected HeLa and Neuro2a cells. Lysates of cells transiently transfected with plasmids were fractionated by <t>SDS-PAGE</t> and analyzed by immunoblot using a mouse monoclonal anti-GFP antibody. α-tubulin was used as an internal control. Asterisks (*) indicate aggregation resolved using stacking gels. Arrowheads indicate full-length SYG/SYGQ-NES. Green arrows indicate predicted size. ( c ) Immunoblot analysis of deletion constructs expressed in 293 T cells. Protein (12 μg/lane) was loaded on a 4% to 20% <t>Tris-glycine</t> gradient gel and detected using an anti-GFP antibody for western blot analysis. Asterisks (*) indicate aggregation resolved using stacking gels. ( d ) Immunoblot analysis of SYG, SYGQ, and SYGQ/N-NES expression in transfected 293 T cells. α-tubulin was used as an internal control. Asterisks (*) indicate aggregation, and arrowheads indicate full-length proteins. ( e ) Neuro2a cell lysates expressed of SYG/SYGQ-NES were separated into RIPA-soluble and -insoluble urea fractions. Full-length proteins (indicated by arrowheads) were detected in only the insoluble fractions.
    Tris Glycine Gel, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 255 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher tris glycine gel
    RNAP containing βG1249D generates holoenzyme with AsiA/σD581BpA, but is defective in generating the crosslink with β’. ( A ) Native <t>Tris-glycine</t> gel. Solutions were assembled with the indicated components. The positions of AsiA, RNAP core, AsiA-RNAP holoenzyme, and σD581BpA are marked. (The bands that migrate faster than AsiA seen in lane 1 are trace contaminants present in the σD581BpA preparation.) ( B ) SDS-PAGE gel showing the products obtained after <t>photocrosslinking.</t> Arrow points to the crosslink between σD581BpA and β’ 80 HRGVICEK 87 identified in Figure 3 .
    Tris Glycine Gel, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 4727 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Bio-Rad tris glycine gels
    Aβ species including oAβ are present in both extracellular and intracellular fractions from young TgCRND8 mouse brains. Levels of human Aβ40 ( A , B ), human/rodent Aβ42 ( C , D ), and oAβ ( E , F ) were measured in the <t>Tris-soluble,</t> extracellular enriched fraction and the <t>RIPA-extracted</t> intracellular vesicle fraction from 2-mo-old (preamyloid deposition) TgCRND8 mouse brains ( n = 10). Identically prepared extracts from wild-type (wt) littermates ( n = 5) were included as controls to validate Aβ measurements in transgenic mice. For every assay, the signal in TgCRND8 transgenic mice was significantly higher than in wild-type mice. ** P
    Tris Glycine Gels, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 1320 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher novex tris glycine gels
    Aβ species including oAβ are present in both extracellular and intracellular fractions from young TgCRND8 mouse brains. Levels of human Aβ40 ( A , B ), human/rodent Aβ42 ( C , D ), and oAβ ( E , F ) were measured in the <t>Tris-soluble,</t> extracellular enriched fraction and the <t>RIPA-extracted</t> intracellular vesicle fraction from 2-mo-old (preamyloid deposition) TgCRND8 mouse brains ( n = 10). Identically prepared extracts from wild-type (wt) littermates ( n = 5) were included as controls to validate Aβ measurements in transgenic mice. For every assay, the signal in TgCRND8 transgenic mice was significantly higher than in wild-type mice. ** P
    Novex Tris Glycine Gels, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 765 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher tris glycine polyacrylamide gels
    Detection of <t>anti-HCV</t> antibodies in HCV RNA and antibody positive human sera with recombinant HCV proteins . 1 μg of each baculovirus-expressed and preparative SDS-PAGE-purified recombinant HCV protein was loaded onto 10–20% <t>Tris-glycine</t> polyacrylamide gradient gels. Core, NS2, NS3, NS4A, NS4B, and NS5A were loaded on one gel, and E1, E2, and NS5B on another gel, respectively. 3 μg of Sf 9 cell extract was also loaded onto one gel as a control. Proteins separated on gels were transferred to nylon membranes, sliced and stained with serially diluted human serum obtained from HCV RNA and antibody positive patients. The following dilutions were used (lane 1) 1:100, (lane 2) 1:500, (lane 3) 1:2500, (lane 4) 1:12500 and 1:62500 (not shown). After incubation with secondary Abs, the bands were visualized by 3-amino-9-ethylcarbazole (AEC). A. An example of highly positive human serum number 36 is shown, B. an example of a weakly positive human serum number 17 is shown.
    Tris Glycine Polyacrylamide Gels, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 674 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher gradient tris glycine gels
    Molecular PrP res typing of atypical scrapie affected tg338 mice. Western blots for PrP res of representative brain extracts following <t>SDS-PAGE</t> using (A) 16.5% <t>tris-glycine</t> and (B-F) 4–20% tris-glycine gradient gels. Immunochemical detection was performed with antibodies P4, Sha31 or 9A2 as indicated. In some instances ( D , E) , samples were deglycosylated with PNGaseF. (F) Atypical scrapie PrP res banding pattern following sample pretreatment with different concentrations of proteinase K (PK, in µg/ml), compared to the PK digestion protocol of the Bio-Rad TeSeE confirmatory Western blot (BR). Identification codes for the atypical scrapie isolates are shown at the top. Controls: cl, classical scrapie affected tg338 mouse; non-inoc, non-inoculated tg 338 mouse; neg s, TSE negative sheep. Molecular mass standards in kDa and loaded tissue equivalents are indicated.
    Gradient Tris Glycine Gels, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 469 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher tris glycine mini gels
    Molecular PrP res typing of atypical scrapie affected tg338 mice. Western blots for PrP res of representative brain extracts following <t>SDS-PAGE</t> using (A) 16.5% <t>tris-glycine</t> and (B-F) 4–20% tris-glycine gradient gels. Immunochemical detection was performed with antibodies P4, Sha31 or 9A2 as indicated. In some instances ( D , E) , samples were deglycosylated with PNGaseF. (F) Atypical scrapie PrP res banding pattern following sample pretreatment with different concentrations of proteinase K (PK, in µg/ml), compared to the PK digestion protocol of the Bio-Rad TeSeE confirmatory Western blot (BR). Identification codes for the atypical scrapie isolates are shown at the top. Controls: cl, classical scrapie affected tg338 mouse; non-inoc, non-inoculated tg 338 mouse; neg s, TSE negative sheep. Molecular mass standards in kDa and loaded tissue equivalents are indicated.
    Tris Glycine Mini Gels, 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 novex tris glycine gel
    Molecular PrP res typing of atypical scrapie affected tg338 mice. Western blots for PrP res of representative brain extracts following <t>SDS-PAGE</t> using (A) 16.5% <t>tris-glycine</t> and (B-F) 4–20% tris-glycine gradient gels. Immunochemical detection was performed with antibodies P4, Sha31 or 9A2 as indicated. In some instances ( D , E) , samples were deglycosylated with PNGaseF. (F) Atypical scrapie PrP res banding pattern following sample pretreatment with different concentrations of proteinase K (PK, in µg/ml), compared to the PK digestion protocol of the Bio-Rad TeSeE confirmatory Western blot (BR). Identification codes for the atypical scrapie isolates are shown at the top. Controls: cl, classical scrapie affected tg338 mouse; non-inoc, non-inoculated tg 338 mouse; neg s, TSE negative sheep. Molecular mass standards in kDa and loaded tissue equivalents are indicated.
    Novex Tris Glycine Gel, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 398 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher tris glycine polyacrylamide gel
    Immunoblot assays and densitometry for V. cholerae virulence factors <t>TcpA</t> and ToxT. Whole-cell extracts (WCE) were prepared, and 8 and 10 μg of total protein for TcpA and ToxT, respectively, was loaded onto a 16% <t>Tris-glycine</t> polyacrylamide gel.
    Tris Glycine Polyacrylamide Gel, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 447 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher polyacrylamide gels
    <t>PER,</t> TIM, and CLK are found in protein complexes containing CK2. Anti-FLAG immunoprecipitation (IP) from nonsonicated head extracts of flies collected at DD1. FLAG-CK2α or FLAG-CK2β was immunoprecipitated from 1 mg of total protein extracts from heads and 50% of the precipitate was subjected to Western blot analysis. We loaded 50 µg of head extracts as input controls. IgG LC indicates the immunoglobulin G light chain used for the precipitation that is well detected by the anti-mouse secondary antibody. Gray and black bars represent subjective day and subjective night, respectively. Experiments were performed at least twice. (A, Left) FLAG-CK2α was immunoprecipitated from tim > FLAG-CkIIα flies and tim-gal4 /+ negative controls (control) in a per + background. IP of CK2α (FLAG) and co-IP of CK2β, TIM, PER, and CLK were visualized by immunoblotting. «*» shows an aspecific band recognized by the anti-PER antibody. (Right) TIM, PER, and CLK immunoblots of the corresponding inputs on per + background. The time “CT10” indicates a mixed population of flies harvested at CT8 and CT12. A CB stained band in the size range of CLK is used as a loading control. TIM was run on a 3–8% <t>Tris-Acetate</t> gel. (B, Left) FLAG-CK2β was immunoprecipitated from tim > FLAG-CkIIß flies and tim-gal4 /+ negative controls (control) in a per + background. IP of CK2ß (with anti-CK2ß) and co-IP of CK2α, TIM, PER, and CLK were visualized by immunoblotting. (Right) TIM, PER, and CLK immunoblots of the corresponding inputs. A CB stained band in the size range of CLK is used as a loading control. (C) FLAG-CK2α or FLAG-CK2β was immunoprecipitated from tim > FLAG-CkIIα ( α ) flies and tim-gal4 /+ negative controls (C) in a per 0 background. co-IP of CLK was visualized by immunoblotting. Input samples and immunoprecipitates were run on the same gel for C and α. (D) Image showing PDF (green), CK2α (magenta), and CLK (blue) fluorescent immunolabeling in small PDF + LNv-s of an adult fly at ZT3. The fourth square is a composite picture of the three stainings. Single optical planes are shown taken by confocal microscopy.
    Polyacrylamide Gels, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 7143 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher novex
    <t>PER,</t> TIM, and CLK are found in protein complexes containing CK2. Anti-FLAG immunoprecipitation (IP) from nonsonicated head extracts of flies collected at DD1. FLAG-CK2α or FLAG-CK2β was immunoprecipitated from 1 mg of total protein extracts from heads and 50% of the precipitate was subjected to Western blot analysis. We loaded 50 µg of head extracts as input controls. IgG LC indicates the immunoglobulin G light chain used for the precipitation that is well detected by the anti-mouse secondary antibody. Gray and black bars represent subjective day and subjective night, respectively. Experiments were performed at least twice. (A, Left) FLAG-CK2α was immunoprecipitated from tim > FLAG-CkIIα flies and tim-gal4 /+ negative controls (control) in a per + background. IP of CK2α (FLAG) and co-IP of CK2β, TIM, PER, and CLK were visualized by immunoblotting. «*» shows an aspecific band recognized by the anti-PER antibody. (Right) TIM, PER, and CLK immunoblots of the corresponding inputs on per + background. The time “CT10” indicates a mixed population of flies harvested at CT8 and CT12. A CB stained band in the size range of CLK is used as a loading control. TIM was run on a 3–8% <t>Tris-Acetate</t> gel. (B, Left) FLAG-CK2β was immunoprecipitated from tim > FLAG-CkIIß flies and tim-gal4 /+ negative controls (control) in a per + background. IP of CK2ß (with anti-CK2ß) and co-IP of CK2α, TIM, PER, and CLK were visualized by immunoblotting. (Right) TIM, PER, and CLK immunoblots of the corresponding inputs. A CB stained band in the size range of CLK is used as a loading control. (C) FLAG-CK2α or FLAG-CK2β was immunoprecipitated from tim > FLAG-CkIIα ( α ) flies and tim-gal4 /+ negative controls (C) in a per 0 background. co-IP of CLK was visualized by immunoblotting. Input samples and immunoprecipitates were run on the same gel for C and α. (D) Image showing PDF (green), CK2α (magenta), and CLK (blue) fluorescent immunolabeling in small PDF + LNv-s of an adult fly at ZT3. The fourth square is a composite picture of the three stainings. Single optical planes are shown taken by confocal microscopy.
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    99
    Millipore tris glycine gels
    <t>PER,</t> TIM, and CLK are found in protein complexes containing CK2. Anti-FLAG immunoprecipitation (IP) from nonsonicated head extracts of flies collected at DD1. FLAG-CK2α or FLAG-CK2β was immunoprecipitated from 1 mg of total protein extracts from heads and 50% of the precipitate was subjected to Western blot analysis. We loaded 50 µg of head extracts as input controls. IgG LC indicates the immunoglobulin G light chain used for the precipitation that is well detected by the anti-mouse secondary antibody. Gray and black bars represent subjective day and subjective night, respectively. Experiments were performed at least twice. (A, Left) FLAG-CK2α was immunoprecipitated from tim > FLAG-CkIIα flies and tim-gal4 /+ negative controls (control) in a per + background. IP of CK2α (FLAG) and co-IP of CK2β, TIM, PER, and CLK were visualized by immunoblotting. «*» shows an aspecific band recognized by the anti-PER antibody. (Right) TIM, PER, and CLK immunoblots of the corresponding inputs on per + background. The time “CT10” indicates a mixed population of flies harvested at CT8 and CT12. A CB stained band in the size range of CLK is used as a loading control. TIM was run on a 3–8% <t>Tris-Acetate</t> gel. (B, Left) FLAG-CK2β was immunoprecipitated from tim > FLAG-CkIIß flies and tim-gal4 /+ negative controls (control) in a per + background. IP of CK2ß (with anti-CK2ß) and co-IP of CK2α, TIM, PER, and CLK were visualized by immunoblotting. (Right) TIM, PER, and CLK immunoblots of the corresponding inputs. A CB stained band in the size range of CLK is used as a loading control. (C) FLAG-CK2α or FLAG-CK2β was immunoprecipitated from tim > FLAG-CkIIα ( α ) flies and tim-gal4 /+ negative controls (C) in a per 0 background. co-IP of CLK was visualized by immunoblotting. Input samples and immunoprecipitates were run on the same gel for C and α. (D) Image showing PDF (green), CK2α (magenta), and CLK (blue) fluorescent immunolabeling in small PDF + LNv-s of an adult fly at ZT3. The fourth square is a composite picture of the three stainings. Single optical planes are shown taken by confocal microscopy.
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    Image Search Results


    Cysteine desulfhydrase activity by in situ staining. MGL activity was monitored in Tris-glycine gels under nondenaturing conditions using l -cysteine as the substrate. Lanes: M, molecular mass markers; 1, purified recombinant His-tagged MGL protein; 2,

    Journal: Applied and Environmental Microbiology

    Article Title: Heterologous Production of Methionine-?-Lyase from Brevibacterium linens in Lactococcus lactis and Formation of Volatile Sulfur Compounds ▿ and Formation of Volatile Sulfur Compounds ▿ †

    doi: 10.1128/AEM.02417-08

    Figure Lengend Snippet: Cysteine desulfhydrase activity by in situ staining. MGL activity was monitored in Tris-glycine gels under nondenaturing conditions using l -cysteine as the substrate. Lanes: M, molecular mass markers; 1, purified recombinant His-tagged MGL protein; 2,

    Article Snippet: The activities of purified His-tagged MGL or CFEs from recombinant L. lactis cultures were monitored using Tris-glycine gels (Invitrogen) run under nondenaturing conditions.

    Techniques: Activity Assay, In Situ, Staining, Purification, Recombinant

    Western blot analysis of WT and apoA-I M in mouse plasma. Seven weeks after AAV injection, apoA-I-null mice were bled and 1 μl of plasma was run on a 4–12% SDS-PAGE Tris-glycine gel. The samples were transferred to nitrocellulose and Western blotted for human apoA-I. Monomeric apoA-I M had an apparent molecular mass of approximately 28 kDa, while dimeric apoA-I M had a molecular mass of approximately 56 kDa. Nonreduced (A) and reduced (B) conditions.

    Journal: Journal of Lipid Research

    Article Title: Structural and functional consequences of the Milano mutation (R173C) in human apolipoprotein A-I

    doi: 10.1194/jlr.M800578-JLR200

    Figure Lengend Snippet: Western blot analysis of WT and apoA-I M in mouse plasma. Seven weeks after AAV injection, apoA-I-null mice were bled and 1 μl of plasma was run on a 4–12% SDS-PAGE Tris-glycine gel. The samples were transferred to nitrocellulose and Western blotted for human apoA-I. Monomeric apoA-I M had an apparent molecular mass of approximately 28 kDa, while dimeric apoA-I M had a molecular mass of approximately 56 kDa. Nonreduced (A) and reduced (B) conditions.

    Article Snippet: Plasma samples (1 μl) were resolved on 4–20% nondenaturing, Tris-glycine gradient gels (Invitrogen) or 4–12% SDS-PAGE Tris-glycine gels (Invitrogen) and transferred to a nitrocellulose membrane.

    Techniques: Western Blot, Injection, Mouse Assay, SDS Page

    Western Blot analysis of RAb DMvIII antibody . A) 30 ug of U87vIII and U87 wild type cell lysate loaded under reducing conditions on 4-20% SDS-PAGE. A) RAb vIII recognizes 145 kDa band which corresponds to the EGFRvIII and 45 kDa unspecific protein band. A) RAb DMvIII recognizes only 145 kDa protein band. B) RAb DMvIII recognizes 170 KDa protein band in 30 ug of A431 cell lysate (cells overexpressing EGFR) and 145 kDa band in 30 ug of HC2 cell lysate under reducing conditions. B) EGFR antibody recognizes equal amount of protein in both HC2 and A431 cell lysate under reducing conditions. C) RAb DMvIII under non reducing conditions detect only 145 kDa protein band whereas RAb vIII still recognizes 145 kDa and 170 kDa protein bands in non reducing conditions. C) Actin antibody under reducing condition shows equal amount of protein loaded in the wells.

    Journal: BMC Biotechnology

    Article Title: Development of an EGFRvIII specific recombinant antibody

    doi: 10.1186/1472-6750-10-72

    Figure Lengend Snippet: Western Blot analysis of RAb DMvIII antibody . A) 30 ug of U87vIII and U87 wild type cell lysate loaded under reducing conditions on 4-20% SDS-PAGE. A) RAb vIII recognizes 145 kDa band which corresponds to the EGFRvIII and 45 kDa unspecific protein band. A) RAb DMvIII recognizes only 145 kDa protein band. B) RAb DMvIII recognizes 170 KDa protein band in 30 ug of A431 cell lysate (cells overexpressing EGFR) and 145 kDa band in 30 ug of HC2 cell lysate under reducing conditions. B) EGFR antibody recognizes equal amount of protein in both HC2 and A431 cell lysate under reducing conditions. C) RAb DMvIII under non reducing conditions detect only 145 kDa protein band whereas RAb vIII still recognizes 145 kDa and 170 kDa protein bands in non reducing conditions. C) Actin antibody under reducing condition shows equal amount of protein loaded in the wells.

    Article Snippet: The proteins were resolved on 4-20% Tris-glycine SDS-PAGE gels (Invitrogen) and transferred to nitrocellulose membrane for immunoblot analysis.

    Techniques: Western Blot, SDS Page

    Expression in cultured mammalian cells of artificial RNA-binding proteins containing PrLDs. ( a ) Schematic diagram showing artificial RNA-binding proteins containing PrLDs, as well as the deletion constructs. All constructs contained a GFP tag at the C-terminus. sPFD: synthetic prion-forming domain; cPFD: control prion-forming domain [exhibiting low degrees of prion propensity 17 ]. ( b ) Immunoblot analysis of SYG/SYGQ-NES expression in transfected HeLa and Neuro2a cells. Lysates of cells transiently transfected with plasmids were fractionated by SDS-PAGE and analyzed by immunoblot using a mouse monoclonal anti-GFP antibody. α-tubulin was used as an internal control. Asterisks (*) indicate aggregation resolved using stacking gels. Arrowheads indicate full-length SYG/SYGQ-NES. Green arrows indicate predicted size. ( c ) Immunoblot analysis of deletion constructs expressed in 293 T cells. Protein (12 μg/lane) was loaded on a 4% to 20% Tris-glycine gradient gel and detected using an anti-GFP antibody for western blot analysis. Asterisks (*) indicate aggregation resolved using stacking gels. ( d ) Immunoblot analysis of SYG, SYGQ, and SYGQ/N-NES expression in transfected 293 T cells. α-tubulin was used as an internal control. Asterisks (*) indicate aggregation, and arrowheads indicate full-length proteins. ( e ) Neuro2a cell lysates expressed of SYG/SYGQ-NES were separated into RIPA-soluble and -insoluble urea fractions. Full-length proteins (indicated by arrowheads) were detected in only the insoluble fractions.

    Journal: Scientific Reports

    Article Title: De novo design of RNA-binding proteins with a prion-like domain related to ALS/FTD proteinopathies

    doi: 10.1038/s41598-017-17209-0

    Figure Lengend Snippet: Expression in cultured mammalian cells of artificial RNA-binding proteins containing PrLDs. ( a ) Schematic diagram showing artificial RNA-binding proteins containing PrLDs, as well as the deletion constructs. All constructs contained a GFP tag at the C-terminus. sPFD: synthetic prion-forming domain; cPFD: control prion-forming domain [exhibiting low degrees of prion propensity 17 ]. ( b ) Immunoblot analysis of SYG/SYGQ-NES expression in transfected HeLa and Neuro2a cells. Lysates of cells transiently transfected with plasmids were fractionated by SDS-PAGE and analyzed by immunoblot using a mouse monoclonal anti-GFP antibody. α-tubulin was used as an internal control. Asterisks (*) indicate aggregation resolved using stacking gels. Arrowheads indicate full-length SYG/SYGQ-NES. Green arrows indicate predicted size. ( c ) Immunoblot analysis of deletion constructs expressed in 293 T cells. Protein (12 μg/lane) was loaded on a 4% to 20% Tris-glycine gradient gel and detected using an anti-GFP antibody for western blot analysis. Asterisks (*) indicate aggregation resolved using stacking gels. ( d ) Immunoblot analysis of SYG, SYGQ, and SYGQ/N-NES expression in transfected 293 T cells. α-tubulin was used as an internal control. Asterisks (*) indicate aggregation, and arrowheads indicate full-length proteins. ( e ) Neuro2a cell lysates expressed of SYG/SYGQ-NES were separated into RIPA-soluble and -insoluble urea fractions. Full-length proteins (indicated by arrowheads) were detected in only the insoluble fractions.

    Article Snippet: Cell lysates were separated using SDS-PAGE with 10% Tris-glycine gel, after which proteins were transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA).

    Techniques: Expressing, Cell Culture, RNA Binding Assay, Construct, Transfection, SDS Page, Western Blot

    RNAP containing βG1249D generates holoenzyme with AsiA/σD581BpA, but is defective in generating the crosslink with β’. ( A ) Native Tris-glycine gel. Solutions were assembled with the indicated components. The positions of AsiA, RNAP core, AsiA-RNAP holoenzyme, and σD581BpA are marked. (The bands that migrate faster than AsiA seen in lane 1 are trace contaminants present in the σD581BpA preparation.) ( B ) SDS-PAGE gel showing the products obtained after photocrosslinking. Arrow points to the crosslink between σD581BpA and β’ 80 HRGVICEK 87 identified in Figure 3 .

    Journal: Nucleic Acids Research

    Article Title: Visualizing the phage T4 activated transcription complex of DNA and E. coli RNA polymerase

    doi: 10.1093/nar/gkw656

    Figure Lengend Snippet: RNAP containing βG1249D generates holoenzyme with AsiA/σD581BpA, but is defective in generating the crosslink with β’. ( A ) Native Tris-glycine gel. Solutions were assembled with the indicated components. The positions of AsiA, RNAP core, AsiA-RNAP holoenzyme, and σD581BpA are marked. (The bands that migrate faster than AsiA seen in lane 1 are trace contaminants present in the σD581BpA preparation.) ( B ) SDS-PAGE gel showing the products obtained after photocrosslinking. Arrow points to the crosslink between σD581BpA and β’ 80 HRGVICEK 87 identified in Figure 3 .

    Article Snippet: A 20 μl aliquot was used for photocrosslinking, and a 5 μl aliquot was applied to a 4–12% Tris-glycine gel (Invitrogen/Thermo Fisher) run in 1× Native Tris-glycine buffer (Invitrogen/Thermo Fisher) and stained in Gel Code (Thermo Fisher) as described ( ).

    Techniques: SDS Page

    SDS-PAGE analysis of native Msg. Msg was purified as described in the methods and then analyzed by SDS-PAGE; the gel was stained with Coomassie Blue. Lane 2 shows Msg (arrow) with an approximate size of ~95 kilodaltons. Molecular weight markers are in lane 1, with their size in kilodaltons indicated on the left.

    Journal: BMC Immunology

    Article Title: Discordant antibody and cellular responses to Pneumocystis major surface glycoprotein variants in mice

    doi: 10.1186/1471-2172-13-39

    Figure Lengend Snippet: SDS-PAGE analysis of native Msg. Msg was purified as described in the methods and then analyzed by SDS-PAGE; the gel was stained with Coomassie Blue. Lane 2 shows Msg (arrow) with an approximate size of ~95 kilodaltons. Molecular weight markers are in lane 1, with their size in kilodaltons indicated on the left.

    Article Snippet: Immunoblot Approximately 34 μg crude P. murina antigen and 0.3 μg native msg antigen were run on a 4-20% Tris-Glycine SDS-PAGE gel (Invitrogen).

    Techniques: SDS Page, Purification, Staining, Molecular Weight

    Aβ species including oAβ are present in both extracellular and intracellular fractions from young TgCRND8 mouse brains. Levels of human Aβ40 ( A , B ), human/rodent Aβ42 ( C , D ), and oAβ ( E , F ) were measured in the Tris-soluble, extracellular enriched fraction and the RIPA-extracted intracellular vesicle fraction from 2-mo-old (preamyloid deposition) TgCRND8 mouse brains ( n = 10). Identically prepared extracts from wild-type (wt) littermates ( n = 5) were included as controls to validate Aβ measurements in transgenic mice. For every assay, the signal in TgCRND8 transgenic mice was significantly higher than in wild-type mice. ** P

    Journal: The FASEB Journal

    Article Title: Intracellular metalloprotease activity controls intraneuronal Aβ aggregation and limits secretion of Aβ via exosomes

    doi: 10.1096/fj.201801319R

    Figure Lengend Snippet: Aβ species including oAβ are present in both extracellular and intracellular fractions from young TgCRND8 mouse brains. Levels of human Aβ40 ( A , B ), human/rodent Aβ42 ( C , D ), and oAβ ( E , F ) were measured in the Tris-soluble, extracellular enriched fraction and the RIPA-extracted intracellular vesicle fraction from 2-mo-old (preamyloid deposition) TgCRND8 mouse brains ( n = 10). Identically prepared extracts from wild-type (wt) littermates ( n = 5) were included as controls to validate Aβ measurements in transgenic mice. For every assay, the signal in TgCRND8 transgenic mice was significantly higher than in wild-type mice. ** P

    Article Snippet: Extracellular vesicle proteins were extracted in RIPA buffer, electrophoresed on 4–20% Tris-glycine gels (Criterion Precast Gel; Bio-Rad, Hercules, CA, USA), and transferred to PVDF membranes (Immobilon; MilliporeSigma).

    Techniques: Transgenic Assay, Mouse Assay

    Intracerebroventricular administration of PA to TgCRND8 and wild-type mice increases extracellular and intracellular Aβ. Aβ species were measured 16 h after icv administration of PA to TgCRND8 mice ( A , B ) and wild-type mice ( C , D ). A , B ) Levels of Tris-soluble extracellular ( A ) and RIPA-soluble intracellular ( B ) Aβ40 and Aβ42 were increased in transgenic mouse brains after PA injection. C , D ) Levels of endogenous rodent Aβ40 and Aβ42 were also increased in extracellular ( C ) and intracellular ( D ) fractions of wild-type mouse brains after PA injection. Intracellular Aβ from wild-type mice was purified and concentrated by solid-phase extraction before measurement by ELISA (for TgCRND8 mice, n = 8 for Aβ40 and n = 10 for Aβ42; for wild-type mice, n = 6 for all groups). **** P

    Journal: The FASEB Journal

    Article Title: Intracellular metalloprotease activity controls intraneuronal Aβ aggregation and limits secretion of Aβ via exosomes

    doi: 10.1096/fj.201801319R

    Figure Lengend Snippet: Intracerebroventricular administration of PA to TgCRND8 and wild-type mice increases extracellular and intracellular Aβ. Aβ species were measured 16 h after icv administration of PA to TgCRND8 mice ( A , B ) and wild-type mice ( C , D ). A , B ) Levels of Tris-soluble extracellular ( A ) and RIPA-soluble intracellular ( B ) Aβ40 and Aβ42 were increased in transgenic mouse brains after PA injection. C , D ) Levels of endogenous rodent Aβ40 and Aβ42 were also increased in extracellular ( C ) and intracellular ( D ) fractions of wild-type mouse brains after PA injection. Intracellular Aβ from wild-type mice was purified and concentrated by solid-phase extraction before measurement by ELISA (for TgCRND8 mice, n = 8 for Aβ40 and n = 10 for Aβ42; for wild-type mice, n = 6 for all groups). **** P

    Article Snippet: Extracellular vesicle proteins were extracted in RIPA buffer, electrophoresed on 4–20% Tris-glycine gels (Criterion Precast Gel; Bio-Rad, Hercules, CA, USA), and transferred to PVDF membranes (Immobilon; MilliporeSigma).

    Techniques: Mouse Assay, Transgenic Assay, Injection, Purification, Enzyme-linked Immunosorbent Assay

    Hydroxylated anthoxanthins alter Aβ oligomer size distribution Oligomers were prepared from Aβ 1-42 (15 μM) in the absence (CONT, control) or presence of 150 μM anthoxanthins flavone (FLA), apigenin (API), luteolin (LUT), kaempferol (KAE), or quercetin (QUE) by dilution from DMSO into 12 mM phosphate (pH 7.4) containing 1 μM NaCl. Following oligomerization (30 min, 25°C), oligomers were stabilized via addition of Tween-20 (0.1%), resolved by SDS-PAGE on either a 4-20% Tris-glycine gel (panel A) or a 16.5% Tris-tricine gel (panel D), transferred to nitrocellulose membrane, and probed with 6E10 antibody. Images are representative of 3-5 independent experiments. Using volumetric analysis in conjunction with the 4-20% Tris-glycine gel images (panel A), oligomer species within size ranges of 100-250 kDa (panel B) and 25-100 kDa (panel C) were quantified. Using band intensity analysis in conjunction with the 16.5% Tris-tricine gel images (panel D), tetramer (panel E), trimer (panel G), and monomer (panel F) species were quantified. Reported results are normalized to the control, shown as a dashed line with a value of 1 and representing no change. Error bars indicate SEM, n=3-5. *p

    Journal: CNS neuroscience & therapeutics

    Article Title: Anthoxanthin polyphenols attenuate Aβ oligomer-induced neuronal responses associated with Alzheimer's disease

    doi: 10.1111/cns.12659

    Figure Lengend Snippet: Hydroxylated anthoxanthins alter Aβ oligomer size distribution Oligomers were prepared from Aβ 1-42 (15 μM) in the absence (CONT, control) or presence of 150 μM anthoxanthins flavone (FLA), apigenin (API), luteolin (LUT), kaempferol (KAE), or quercetin (QUE) by dilution from DMSO into 12 mM phosphate (pH 7.4) containing 1 μM NaCl. Following oligomerization (30 min, 25°C), oligomers were stabilized via addition of Tween-20 (0.1%), resolved by SDS-PAGE on either a 4-20% Tris-glycine gel (panel A) or a 16.5% Tris-tricine gel (panel D), transferred to nitrocellulose membrane, and probed with 6E10 antibody. Images are representative of 3-5 independent experiments. Using volumetric analysis in conjunction with the 4-20% Tris-glycine gel images (panel A), oligomer species within size ranges of 100-250 kDa (panel B) and 25-100 kDa (panel C) were quantified. Using band intensity analysis in conjunction with the 16.5% Tris-tricine gel images (panel D), tetramer (panel E), trimer (panel G), and monomer (panel F) species were quantified. Reported results are normalized to the control, shown as a dashed line with a value of 1 and representing no change. Error bars indicate SEM, n=3-5. *p

    Article Snippet: For oligomers 25-250 kDa in size, stabilized oligomers were separated on a 4-20% Tris-glycine gel (Bio-rad, Hercules, CA); for monomer, trimer, and tetramer, stabilized oligomers were separated on a 16.5% Tris-tricine gel (Bio-rad).

    Techniques: SDS Page

    Cultures of primary human and mouse bone marrow stroma produce microvesicles containing FGF2. Conditioned medium was collected from cultured human bone marrow stroma. Samples were ultracentrifuged and pellets were lysed with 78 μL of Cell Lysis Buffer (Cell Signaling Technologies Inc, Danvers, MA) containing a Complete Mini Protease Inhibitor Cocktail Tablet, Phosphatase Inhibitor Cocktail 2, and Phenylmethanesulfonyl Fluoride (PMSF) solution (Sigma-Aldrich Inc, St Louis, MO) and clarified by centrifugation at 14,000 g, 4°C for 15 min. All samples were loaded on NuPAGE 4–12% Bis-Tris gradient gels, ran in MES buffer (Thermo Fisher Scientific Inc, Waltham, MA), transferred on Immobilon-FL PVDF membranes (Millipore Inc, Billerica, MA), and blocked overnight at 4°C. Following overnight incubation, membranes were incubated with the following primary antibodies: anti-FGFR1, anti-FGF2, anti-CD63, anti-CD9, and anti-actin ( Supplementary file 1 ) overnight at 4°C. The following day membranes were washed and probed with fluorescent IRDye 800CW goat anti-rabbit IgG and IRDye 680RD Goat anti-mouse IgG antibodies ( Supplementary file 1 ). The membranes were imaged with the Odyssey Infrared Imaging System (LI-COR Biosciences).

    Journal: eLife

    Article Title: FGF2-FGFR1 signaling regulates release of Leukemia-Protective exosomes from bone marrow stromal cells

    doi: 10.7554/eLife.40033

    Figure Lengend Snippet: Cultures of primary human and mouse bone marrow stroma produce microvesicles containing FGF2. Conditioned medium was collected from cultured human bone marrow stroma. Samples were ultracentrifuged and pellets were lysed with 78 μL of Cell Lysis Buffer (Cell Signaling Technologies Inc, Danvers, MA) containing a Complete Mini Protease Inhibitor Cocktail Tablet, Phosphatase Inhibitor Cocktail 2, and Phenylmethanesulfonyl Fluoride (PMSF) solution (Sigma-Aldrich Inc, St Louis, MO) and clarified by centrifugation at 14,000 g, 4°C for 15 min. All samples were loaded on NuPAGE 4–12% Bis-Tris gradient gels, ran in MES buffer (Thermo Fisher Scientific Inc, Waltham, MA), transferred on Immobilon-FL PVDF membranes (Millipore Inc, Billerica, MA), and blocked overnight at 4°C. Following overnight incubation, membranes were incubated with the following primary antibodies: anti-FGFR1, anti-FGF2, anti-CD63, anti-CD9, and anti-actin ( Supplementary file 1 ) overnight at 4°C. The following day membranes were washed and probed with fluorescent IRDye 800CW goat anti-rabbit IgG and IRDye 680RD Goat anti-mouse IgG antibodies ( Supplementary file 1 ). The membranes were imaged with the Odyssey Infrared Imaging System (LI-COR Biosciences).

    Article Snippet: Proteins were fractionated on 4–15% Tris-glycine polyacrylamide gels (Criterion gels, Bio-Rad), transferred to PVDF membranes, and probed with antibodies: FGFR1, fibronectin (Cell Signaling, Danvers, MA, USA); CD9, FGF2, calreticulin, tsg101 (Santa Cruz Biotechnology, Dallas, TX, USA), CD63 (Abcam, Boston, MA, USA), and actin (MAB1501, Millipore, Burlington, MA, USA).

    Techniques: Cell Culture, Lysis, Protease Inhibitor, Centrifugation, Incubation, Imaging

    Y. pseudotuberculosis secretes MVs and the proteomes differ based on the strain. ( A ) TEM image showing MVs (arrows) budding off of Y. pseudotuberculosis . ( B , C ) TEM images of MVs purified from cultures of Y. pseudotuberculosis ATCC 29833 ( B ) and YPIII ( C ). ( D , E ) The proteomes of the two MVs differed based upon SDS-PAGE analyses ( D ) and LC-MS-MS ( E ). ( D ) Distinct bands can be seen in the respective MVs, hinting at differences in their proteomes. The full gel is shown in Fig. S1 . ( E ) The LC-MS-MS analyses found a high degree of similarity in the MV proteomes but also some significant difference, particularly in the outer membrane fraction.

    Journal: Scientific Reports

    Article Title: The Cytotoxic Necrotizing Factor of Yersinia pseudotuberculosis (CNFy) is Carried on Extracellular Membrane Vesicles to Host Cells

    doi: 10.1038/s41598-018-32530-y

    Figure Lengend Snippet: Y. pseudotuberculosis secretes MVs and the proteomes differ based on the strain. ( A ) TEM image showing MVs (arrows) budding off of Y. pseudotuberculosis . ( B , C ) TEM images of MVs purified from cultures of Y. pseudotuberculosis ATCC 29833 ( B ) and YPIII ( C ). ( D , E ) The proteomes of the two MVs differed based upon SDS-PAGE analyses ( D ) and LC-MS-MS ( E ). ( D ) Distinct bands can be seen in the respective MVs, hinting at differences in their proteomes. The full gel is shown in Fig. S1 . ( E ) The LC-MS-MS analyses found a high degree of similarity in the MV proteomes but also some significant difference, particularly in the outer membrane fraction.

    Article Snippet: The protein content in each fraction was examined by 10% Tris-glycine SDS-PAGE gels (Biorad, USA).

    Techniques: Transmission Electron Microscopy, Purification, SDS Page, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    Detection of anti-HCV antibodies in HCV RNA and antibody positive human sera with recombinant HCV proteins . 1 μg of each baculovirus-expressed and preparative SDS-PAGE-purified recombinant HCV protein was loaded onto 10–20% Tris-glycine polyacrylamide gradient gels. Core, NS2, NS3, NS4A, NS4B, and NS5A were loaded on one gel, and E1, E2, and NS5B on another gel, respectively. 3 μg of Sf 9 cell extract was also loaded onto one gel as a control. Proteins separated on gels were transferred to nylon membranes, sliced and stained with serially diluted human serum obtained from HCV RNA and antibody positive patients. The following dilutions were used (lane 1) 1:100, (lane 2) 1:500, (lane 3) 1:2500, (lane 4) 1:12500 and 1:62500 (not shown). After incubation with secondary Abs, the bands were visualized by 3-amino-9-ethylcarbazole (AEC). A. An example of highly positive human serum number 36 is shown, B. an example of a weakly positive human serum number 17 is shown.

    Journal: Virology Journal

    Article Title: Hepatitis C virus core, NS3, NS4B and NS5A are the major immunogenic proteins in humoral immunity in chronic HCV infection

    doi: 10.1186/1743-422X-6-84

    Figure Lengend Snippet: Detection of anti-HCV antibodies in HCV RNA and antibody positive human sera with recombinant HCV proteins . 1 μg of each baculovirus-expressed and preparative SDS-PAGE-purified recombinant HCV protein was loaded onto 10–20% Tris-glycine polyacrylamide gradient gels. Core, NS2, NS3, NS4A, NS4B, and NS5A were loaded on one gel, and E1, E2, and NS5B on another gel, respectively. 3 μg of Sf 9 cell extract was also loaded onto one gel as a control. Proteins separated on gels were transferred to nylon membranes, sliced and stained with serially diluted human serum obtained from HCV RNA and antibody positive patients. The following dilutions were used (lane 1) 1:100, (lane 2) 1:500, (lane 3) 1:2500, (lane 4) 1:12500 and 1:62500 (not shown). After incubation with secondary Abs, the bands were visualized by 3-amino-9-ethylcarbazole (AEC). A. An example of highly positive human serum number 36 is shown, B. an example of a weakly positive human serum number 17 is shown.

    Article Snippet: For Western blot analysis 1 μg of each purified HCV protein was loaded onto two Novex pre-cast, preparative 10–20% Tris-glycine polyacrylamide gels (Invitrogen Corp., Carlsbad, CA).

    Techniques: Recombinant, SDS Page, Purification, Staining, Incubation

    Molecular PrP res typing of atypical scrapie affected tg338 mice. Western blots for PrP res of representative brain extracts following SDS-PAGE using (A) 16.5% tris-glycine and (B-F) 4–20% tris-glycine gradient gels. Immunochemical detection was performed with antibodies P4, Sha31 or 9A2 as indicated. In some instances ( D , E) , samples were deglycosylated with PNGaseF. (F) Atypical scrapie PrP res banding pattern following sample pretreatment with different concentrations of proteinase K (PK, in µg/ml), compared to the PK digestion protocol of the Bio-Rad TeSeE confirmatory Western blot (BR). Identification codes for the atypical scrapie isolates are shown at the top. Controls: cl, classical scrapie affected tg338 mouse; non-inoc, non-inoculated tg 338 mouse; neg s, TSE negative sheep. Molecular mass standards in kDa and loaded tissue equivalents are indicated.

    Journal: PLoS ONE

    Article Title: Atypical Scrapie Isolates Involve a Uniform Prion Species with a Complex Molecular Signature

    doi: 10.1371/journal.pone.0027510

    Figure Lengend Snippet: Molecular PrP res typing of atypical scrapie affected tg338 mice. Western blots for PrP res of representative brain extracts following SDS-PAGE using (A) 16.5% tris-glycine and (B-F) 4–20% tris-glycine gradient gels. Immunochemical detection was performed with antibodies P4, Sha31 or 9A2 as indicated. In some instances ( D , E) , samples were deglycosylated with PNGaseF. (F) Atypical scrapie PrP res banding pattern following sample pretreatment with different concentrations of proteinase K (PK, in µg/ml), compared to the PK digestion protocol of the Bio-Rad TeSeE confirmatory Western blot (BR). Identification codes for the atypical scrapie isolates are shown at the top. Controls: cl, classical scrapie affected tg338 mouse; non-inoc, non-inoculated tg 338 mouse; neg s, TSE negative sheep. Molecular mass standards in kDa and loaded tissue equivalents are indicated.

    Article Snippet: PrPres fragments were separated by SDS-PAGE in hand-cast 16.5% tris-glycine gels or in pre-cast 4-20% gradient tris-glycine gels (Invitrogen, EC 6028), transferred to PVDF membranes (Bio-Rad, 162–0177), and blocked with 1% casein in PBS (Bio-Rad, 161–0783).

    Techniques: Mouse Assay, Western Blot, SDS Page

    Immunoblot assays and densitometry for V. cholerae virulence factors TcpA and ToxT. Whole-cell extracts (WCE) were prepared, and 8 and 10 μg of total protein for TcpA and ToxT, respectively, was loaded onto a 16% Tris-glycine polyacrylamide gel.

    Journal: Journal of Clinical Microbiology

    Article Title: Characterization of Vibrio cholerae O1 El Tor Biotype Variant Clinical Isolates from Bangladesh and Haiti, Including a Molecular Genetic Analysis of Virulence Genes ▿

    doi: 10.1128/JCM.01286-11

    Figure Lengend Snippet: Immunoblot assays and densitometry for V. cholerae virulence factors TcpA and ToxT. Whole-cell extracts (WCE) were prepared, and 8 and 10 μg of total protein for TcpA and ToxT, respectively, was loaded onto a 16% Tris-glycine polyacrylamide gel.

    Article Snippet: AKI-inducing conditions involve incubation without aeration for the first 3.5 h, followed by aeration for the next 4 h. WCE protein concentrations were determined, and either 10 (for ToxT) or 8 μg (for TcpA) of WCE was run on a 16% Tris-glycine polyacrylamide gel (Invitrogen, Carlsbad, CA), transferred to nitrocellulose, probed with anti-ToxT or anti-TcpA, and visualized using the ECL (enhanced chemiluminescence) detection system (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom).

    Techniques:

    PER, TIM, and CLK are found in protein complexes containing CK2. Anti-FLAG immunoprecipitation (IP) from nonsonicated head extracts of flies collected at DD1. FLAG-CK2α or FLAG-CK2β was immunoprecipitated from 1 mg of total protein extracts from heads and 50% of the precipitate was subjected to Western blot analysis. We loaded 50 µg of head extracts as input controls. IgG LC indicates the immunoglobulin G light chain used for the precipitation that is well detected by the anti-mouse secondary antibody. Gray and black bars represent subjective day and subjective night, respectively. Experiments were performed at least twice. (A, Left) FLAG-CK2α was immunoprecipitated from tim > FLAG-CkIIα flies and tim-gal4 /+ negative controls (control) in a per + background. IP of CK2α (FLAG) and co-IP of CK2β, TIM, PER, and CLK were visualized by immunoblotting. «*» shows an aspecific band recognized by the anti-PER antibody. (Right) TIM, PER, and CLK immunoblots of the corresponding inputs on per + background. The time “CT10” indicates a mixed population of flies harvested at CT8 and CT12. A CB stained band in the size range of CLK is used as a loading control. TIM was run on a 3–8% Tris-Acetate gel. (B, Left) FLAG-CK2β was immunoprecipitated from tim > FLAG-CkIIß flies and tim-gal4 /+ negative controls (control) in a per + background. IP of CK2ß (with anti-CK2ß) and co-IP of CK2α, TIM, PER, and CLK were visualized by immunoblotting. (Right) TIM, PER, and CLK immunoblots of the corresponding inputs. A CB stained band in the size range of CLK is used as a loading control. (C) FLAG-CK2α or FLAG-CK2β was immunoprecipitated from tim > FLAG-CkIIα ( α ) flies and tim-gal4 /+ negative controls (C) in a per 0 background. co-IP of CLK was visualized by immunoblotting. Input samples and immunoprecipitates were run on the same gel for C and α. (D) Image showing PDF (green), CK2α (magenta), and CLK (blue) fluorescent immunolabeling in small PDF + LNv-s of an adult fly at ZT3. The fourth square is a composite picture of the three stainings. Single optical planes are shown taken by confocal microscopy.

    Journal: PLoS Biology

    Article Title: The CK2 Kinase Stabilizes CLOCK and Represses Its Activity in the Drosophila Circadian Oscillator

    doi: 10.1371/journal.pbio.1001645

    Figure Lengend Snippet: PER, TIM, and CLK are found in protein complexes containing CK2. Anti-FLAG immunoprecipitation (IP) from nonsonicated head extracts of flies collected at DD1. FLAG-CK2α or FLAG-CK2β was immunoprecipitated from 1 mg of total protein extracts from heads and 50% of the precipitate was subjected to Western blot analysis. We loaded 50 µg of head extracts as input controls. IgG LC indicates the immunoglobulin G light chain used for the precipitation that is well detected by the anti-mouse secondary antibody. Gray and black bars represent subjective day and subjective night, respectively. Experiments were performed at least twice. (A, Left) FLAG-CK2α was immunoprecipitated from tim > FLAG-CkIIα flies and tim-gal4 /+ negative controls (control) in a per + background. IP of CK2α (FLAG) and co-IP of CK2β, TIM, PER, and CLK were visualized by immunoblotting. «*» shows an aspecific band recognized by the anti-PER antibody. (Right) TIM, PER, and CLK immunoblots of the corresponding inputs on per + background. The time “CT10” indicates a mixed population of flies harvested at CT8 and CT12. A CB stained band in the size range of CLK is used as a loading control. TIM was run on a 3–8% Tris-Acetate gel. (B, Left) FLAG-CK2β was immunoprecipitated from tim > FLAG-CkIIß flies and tim-gal4 /+ negative controls (control) in a per + background. IP of CK2ß (with anti-CK2ß) and co-IP of CK2α, TIM, PER, and CLK were visualized by immunoblotting. (Right) TIM, PER, and CLK immunoblots of the corresponding inputs. A CB stained band in the size range of CLK is used as a loading control. (C) FLAG-CK2α or FLAG-CK2β was immunoprecipitated from tim > FLAG-CkIIα ( α ) flies and tim-gal4 /+ negative controls (C) in a per 0 background. co-IP of CLK was visualized by immunoblotting. Input samples and immunoprecipitates were run on the same gel for C and α. (D) Image showing PDF (green), CK2α (magenta), and CLK (blue) fluorescent immunolabeling in small PDF + LNv-s of an adult fly at ZT3. The fourth square is a composite picture of the three stainings. Single optical planes are shown taken by confocal microscopy.

    Article Snippet: We loaded 50 µg total protein on Novex 4% Tris-Glycine precast gels (Life Technologies) for PER, TIM, and CLK immunoblotting, except specifically indicated.

    Techniques: Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay, Staining, Immunolabeling, Confocal Microscopy

    CK2α overexpression induces CLK hyperphosphorylation in the presence of PER. (A, B) Western blot of nonsonicated head extracts from flies collected at DD1. Samples were run on 3–8% Tris-Acetate gels in order to better resolve hyperphosphorylated CLK and TIM forms. Gray and black bars represent subjective day and subjective night, respectively. At least two independent experiments were performed for each blot. (A, Top) Comparison between tim > CkIIα and tim-gal4/+ controls in a per + background, for TIM, PER, and CLK proteins. (Bottom) Two independent experiments as above were quantified for CLK abundance, and the mean values are plotted. Error bars stand for the difference of the respective values from each experiment and their mean. The value of w; tim-gal4/+ at CT0 was normalized to 100. (B) Comparison between per 0 ; tim > CkIIα and per 0 ; tim-gal4/+ controls for CLK. (C) CK2α phosphorylates CLK in vitro . (Top) Wild-type CLK was translated with a N-terminal 6-histidine fusion tag in vitro , affinity purified either in the absence or presence of PER or TIM, and subjected to phosphorylation assays by incubation with γ– 32 P-ATP either in the absence (−) or presence (+) of CK2α. Intensity of incorporated 32 P-phosphate into CLK ( 32 P) was analyzed by autoradiography and total CLK protein levels (CLK) were determined by Western blot analysis. The arrow indicates the position of phosphorylated CLK. (Bottom) Quantification of CLK-incorporated 32 P-phosphate after normalization towards total CLK protein levels. Average CLK phosphorylation from at least three experiments ± s.e.m. are shown in the figure with wild-type CLK set to 100.

    Journal: PLoS Biology

    Article Title: The CK2 Kinase Stabilizes CLOCK and Represses Its Activity in the Drosophila Circadian Oscillator

    doi: 10.1371/journal.pbio.1001645

    Figure Lengend Snippet: CK2α overexpression induces CLK hyperphosphorylation in the presence of PER. (A, B) Western blot of nonsonicated head extracts from flies collected at DD1. Samples were run on 3–8% Tris-Acetate gels in order to better resolve hyperphosphorylated CLK and TIM forms. Gray and black bars represent subjective day and subjective night, respectively. At least two independent experiments were performed for each blot. (A, Top) Comparison between tim > CkIIα and tim-gal4/+ controls in a per + background, for TIM, PER, and CLK proteins. (Bottom) Two independent experiments as above were quantified for CLK abundance, and the mean values are plotted. Error bars stand for the difference of the respective values from each experiment and their mean. The value of w; tim-gal4/+ at CT0 was normalized to 100. (B) Comparison between per 0 ; tim > CkIIα and per 0 ; tim-gal4/+ controls for CLK. (C) CK2α phosphorylates CLK in vitro . (Top) Wild-type CLK was translated with a N-terminal 6-histidine fusion tag in vitro , affinity purified either in the absence or presence of PER or TIM, and subjected to phosphorylation assays by incubation with γ– 32 P-ATP either in the absence (−) or presence (+) of CK2α. Intensity of incorporated 32 P-phosphate into CLK ( 32 P) was analyzed by autoradiography and total CLK protein levels (CLK) were determined by Western blot analysis. The arrow indicates the position of phosphorylated CLK. (Bottom) Quantification of CLK-incorporated 32 P-phosphate after normalization towards total CLK protein levels. Average CLK phosphorylation from at least three experiments ± s.e.m. are shown in the figure with wild-type CLK set to 100.

    Article Snippet: We loaded 50 µg total protein on Novex 4% Tris-Glycine precast gels (Life Technologies) for PER, TIM, and CLK immunoblotting, except specifically indicated.

    Techniques: Over Expression, Western Blot, In Vitro, Affinity Purification, Incubation, Autoradiography