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  • 99
    Qiagen ni nta his bind superflow column
    Ni Nta His Bind Superflow Column, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher cdna size fractionation columns
    Summary of cDNAs recovered from DENV-resistant Huh7.5 cell clones. Fifty-two colonies of clonal Huh7.5 cells that survived a chellenge infection of DENV-2 were infected with DENV at an MOI of 1, and culture supernatants of infected cells were subjected to plaque assay. <t>mRNA</t> were isolated from cells whose supernatant formed 10 or less DENV plaques less (DENV-resistant clones, total 32 clones), and <t>cDNA</t> inserts expressing in the clones were analyzed by sequencing. More than 50 plaques were formed with a supernatant of control protein (BAP)-expressing Huh7.5 cells.
    Cdna Size Fractionation Columns, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 71 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Millipore mini column fractionation kit
    Summary of cDNAs recovered from DENV-resistant Huh7.5 cell clones. Fifty-two colonies of clonal Huh7.5 cells that survived a chellenge infection of DENV-2 were infected with DENV at an MOI of 1, and culture supernatants of infected cells were subjected to plaque assay. <t>mRNA</t> were isolated from cells whose supernatant formed 10 or less DENV plaques less (DENV-resistant clones, total 32 clones), and <t>cDNA</t> inserts expressing in the clones were analyzed by sequencing. More than 50 plaques were formed with a supernatant of control protein (BAP)-expressing Huh7.5 cells.
    Mini Column Fractionation Kit, supplied by Millipore, used in various techniques. Bioz Stars score: 87/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore column chromatography fractions
    Summary of cDNAs recovered from DENV-resistant Huh7.5 cell clones. Fifty-two colonies of clonal Huh7.5 cells that survived a chellenge infection of DENV-2 were infected with DENV at an MOI of 1, and culture supernatants of infected cells were subjected to plaque assay. <t>mRNA</t> were isolated from cells whose supernatant formed 10 or less DENV plaques less (DENV-resistant clones, total 32 clones), and <t>cDNA</t> inserts expressing in the clones were analyzed by sequencing. More than 50 plaques were formed with a supernatant of control protein (BAP)-expressing Huh7.5 cells.
    Column Chromatography Fractions, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 234 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    GE Healthcare column dialyzed fractions
    Summary of cDNAs recovered from DENV-resistant Huh7.5 cell clones. Fifty-two colonies of clonal Huh7.5 cells that survived a chellenge infection of DENV-2 were infected with DENV at an MOI of 1, and culture supernatants of infected cells were subjected to plaque assay. <t>mRNA</t> were isolated from cells whose supernatant formed 10 or less DENV plaques less (DENV-resistant clones, total 32 clones), and <t>cDNA</t> inserts expressing in the clones were analyzed by sequencing. More than 50 plaques were formed with a supernatant of control protein (BAP)-expressing Huh7.5 cells.
    Column Dialyzed Fractions, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Becton Dickinson pre column fractions
    Summary of cDNAs recovered from DENV-resistant Huh7.5 cell clones. Fifty-two colonies of clonal Huh7.5 cells that survived a chellenge infection of DENV-2 were infected with DENV at an MOI of 1, and culture supernatants of infected cells were subjected to plaque assay. <t>mRNA</t> were isolated from cells whose supernatant formed 10 or less DENV plaques less (DENV-resistant clones, total 32 clones), and <t>cDNA</t> inserts expressing in the clones were analyzed by sequencing. More than 50 plaques were formed with a supernatant of control protein (BAP)-expressing Huh7.5 cells.
    Pre Column Fractions, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 86/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson chromaspin 200 size fractionation column
    Summary of cDNAs recovered from DENV-resistant Huh7.5 cell clones. Fifty-two colonies of clonal Huh7.5 cells that survived a chellenge infection of DENV-2 were infected with DENV at an MOI of 1, and culture supernatants of infected cells were subjected to plaque assay. <t>mRNA</t> were isolated from cells whose supernatant formed 10 or less DENV plaques less (DENV-resistant clones, total 32 clones), and <t>cDNA</t> inserts expressing in the clones were analyzed by sequencing. More than 50 plaques were formed with a supernatant of control protein (BAP)-expressing Huh7.5 cells.
    Chromaspin 200 Size Fractionation Column, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Sartorius AG sartorius fractionation columns
    Summary of cDNAs recovered from DENV-resistant Huh7.5 cell clones. Fifty-two colonies of clonal Huh7.5 cells that survived a chellenge infection of DENV-2 were infected with DENV at an MOI of 1, and culture supernatants of infected cells were subjected to plaque assay. <t>mRNA</t> were isolated from cells whose supernatant formed 10 or less DENV plaques less (DENV-resistant clones, total 32 clones), and <t>cDNA</t> inserts expressing in the clones were analyzed by sequencing. More than 50 plaques were formed with a supernatant of control protein (BAP)-expressing Huh7.5 cells.
    Sartorius Fractionation Columns, supplied by Sartorius AG, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Phenomenex ultremex scx fractionation column
    Summary of cDNAs recovered from DENV-resistant Huh7.5 cell clones. Fifty-two colonies of clonal Huh7.5 cells that survived a chellenge infection of DENV-2 were infected with DENV at an MOI of 1, and culture supernatants of infected cells were subjected to plaque assay. <t>mRNA</t> were isolated from cells whose supernatant formed 10 or less DENV plaques less (DENV-resistant clones, total 32 clones), and <t>cDNA</t> inserts expressing in the clones were analyzed by sequencing. More than 50 plaques were formed with a supernatant of control protein (BAP)-expressing Huh7.5 cells.
    Ultremex Scx Fractionation Column, supplied by Phenomenex, used in various techniques. Bioz Stars score: 95/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Kontes Glass column chromatography fractionation
    Summary of cDNAs recovered from DENV-resistant Huh7.5 cell clones. Fifty-two colonies of clonal Huh7.5 cells that survived a chellenge infection of DENV-2 were infected with DENV at an MOI of 1, and culture supernatants of infected cells were subjected to plaque assay. <t>mRNA</t> were isolated from cells whose supernatant formed 10 or less DENV plaques less (DENV-resistant clones, total 32 clones), and <t>cDNA</t> inserts expressing in the clones were analyzed by sequencing. More than 50 plaques were formed with a supernatant of control protein (BAP)-expressing Huh7.5 cells.
    Column Chromatography Fractionation, supplied by Kontes Glass, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Millipore microcon 50 size fraction spin column
    Summary of cDNAs recovered from DENV-resistant Huh7.5 cell clones. Fifty-two colonies of clonal Huh7.5 cells that survived a chellenge infection of DENV-2 were infected with DENV at an MOI of 1, and culture supernatants of infected cells were subjected to plaque assay. <t>mRNA</t> were isolated from cells whose supernatant formed 10 or less DENV plaques less (DENV-resistant clones, total 32 clones), and <t>cDNA</t> inserts expressing in the clones were analyzed by sequencing. More than 50 plaques were formed with a supernatant of control protein (BAP)-expressing Huh7.5 cells.
    Microcon 50 Size Fraction Spin Column, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Miltenyi Biotec column bound cd11b fraction
    IL4 gene therapy induces M2 markers and IL6 expression in CNS-infiltrating <t>CD11b</t> + cells in vivo. mRNA levels of IL6, IL-1β, and YM1 were measured by real-time RT-PCR in CD11b + cells recovered from the CNS of EAE mice treated intracisternally with an IL4-expressing (IL4) or a GFP-expressing (GFP) lentivirus. IL4 gene therapy induces a decrease of IL-1β ( a ), and an increase of IL6 ( b ) and Ym1 ( c ), as compared to control-treated mice. n = 3 for each group. Gapdh has been used as a housekeeping gene. Data are shown as arbitrary units (AU ± standard deviation). * P
    Column Bound Cd11b Fraction, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 91/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare sp sepharose column fractions
    IL4 gene therapy induces M2 markers and IL6 expression in CNS-infiltrating <t>CD11b</t> + cells in vivo. mRNA levels of IL6, IL-1β, and YM1 were measured by real-time RT-PCR in CD11b + cells recovered from the CNS of EAE mice treated intracisternally with an IL4-expressing (IL4) or a GFP-expressing (GFP) lentivirus. IL4 gene therapy induces a decrease of IL-1β ( a ), and an increase of IL6 ( b ) and Ym1 ( c ), as compared to control-treated mice. n = 3 for each group. Gapdh has been used as a housekeeping gene. Data are shown as arbitrary units (AU ± standard deviation). * P
    Sp Sepharose Column Fractions, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 86/100, based on 349 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa cdna size fractionation columns
    IL4 gene therapy induces M2 markers and IL6 expression in CNS-infiltrating <t>CD11b</t> + cells in vivo. mRNA levels of IL6, IL-1β, and YM1 were measured by real-time RT-PCR in CD11b + cells recovered from the CNS of EAE mice treated intracisternally with an IL4-expressing (IL4) or a GFP-expressing (GFP) lentivirus. IL4 gene therapy induces a decrease of IL-1β ( a ), and an increase of IL6 ( b ) and Ym1 ( c ), as compared to control-treated mice. n = 3 for each group. Gapdh has been used as a housekeeping gene. Data are shown as arbitrary units (AU ± standard deviation). * P
    Cdna Size Fractionation Columns, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Millipore silica gel fractionation column
    IL4 gene therapy induces M2 markers and IL6 expression in CNS-infiltrating <t>CD11b</t> + cells in vivo. mRNA levels of IL6, IL-1β, and YM1 were measured by real-time RT-PCR in CD11b + cells recovered from the CNS of EAE mice treated intracisternally with an IL4-expressing (IL4) or a GFP-expressing (GFP) lentivirus. IL4 gene therapy induces a decrease of IL-1β ( a ), and an increase of IL6 ( b ) and Ym1 ( c ), as compared to control-treated mice. n = 3 for each group. Gapdh has been used as a housekeeping gene. Data are shown as arbitrary units (AU ± standard deviation). * P
    Silica Gel Fractionation Column, supplied by Millipore, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore cl 2b sepharose size fractionation column
    IL4 gene therapy induces M2 markers and IL6 expression in CNS-infiltrating <t>CD11b</t> + cells in vivo. mRNA levels of IL6, IL-1β, and YM1 were measured by real-time RT-PCR in CD11b + cells recovered from the CNS of EAE mice treated intracisternally with an IL4-expressing (IL4) or a GFP-expressing (GFP) lentivirus. IL4 gene therapy induces a decrease of IL-1β ( a ), and an increase of IL6 ( b ) and Ym1 ( c ), as compared to control-treated mice. n = 3 for each group. Gapdh has been used as a housekeeping gene. Data are shown as arbitrary units (AU ± standard deviation). * P
    Cl 2b Sepharose Size Fractionation Column, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Miltenyi Biotec non labeled monocyte enriched fraction
    IL4 gene therapy induces M2 markers and IL6 expression in CNS-infiltrating <t>CD11b</t> + cells in vivo. mRNA levels of IL6, IL-1β, and YM1 were measured by real-time RT-PCR in CD11b + cells recovered from the CNS of EAE mice treated intracisternally with an IL4-expressing (IL4) or a GFP-expressing (GFP) lentivirus. IL4 gene therapy induces a decrease of IL-1β ( a ), and an increase of IL6 ( b ) and Ym1 ( c ), as compared to control-treated mice. n = 3 for each group. Gapdh has been used as a housekeeping gene. Data are shown as arbitrary units (AU ± standard deviation). * P
    Non Labeled Monocyte Enriched Fraction, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 99/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Agilent technologies zorbax 300sb c18 analytical fractions column
    IL4 gene therapy induces M2 markers and IL6 expression in CNS-infiltrating <t>CD11b</t> + cells in vivo. mRNA levels of IL6, IL-1β, and YM1 were measured by real-time RT-PCR in CD11b + cells recovered from the CNS of EAE mice treated intracisternally with an IL4-expressing (IL4) or a GFP-expressing (GFP) lentivirus. IL4 gene therapy induces a decrease of IL-1β ( a ), and an increase of IL6 ( b ) and Ym1 ( c ), as compared to control-treated mice. n = 3 for each group. Gapdh has been used as a housekeeping gene. Data are shown as arbitrary units (AU ± standard deviation). * P
    Zorbax 300sb C18 Analytical Fractions Column, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Millipore lh 20 column fractionation the cr extract powder
    IL4 gene therapy induces M2 markers and IL6 expression in CNS-infiltrating <t>CD11b</t> + cells in vivo. mRNA levels of IL6, IL-1β, and YM1 were measured by real-time RT-PCR in CD11b + cells recovered from the CNS of EAE mice treated intracisternally with an IL4-expressing (IL4) or a GFP-expressing (GFP) lentivirus. IL4 gene therapy induces a decrease of IL-1β ( a ), and an increase of IL6 ( b ) and Ym1 ( c ), as compared to control-treated mice. n = 3 for each group. Gapdh has been used as a housekeeping gene. Data are shown as arbitrary units (AU ± standard deviation). * P
    Lh 20 Column Fractionation The Cr Extract Powder, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Bio-Rad reverse phase hplc fractions
    Western blot analysis of type IX collagen CB-peptides from spondylolisthesis disc with Trp3 polymorphism (D9341). The collagen CB-peptides were partially resolved by reverse-phase <t>HPLC,</t> column fractions were run on <t>SDS-</t> 12.5% PAGE and then transblotted
    Reverse Phase Hplc Fractions, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Miltenyi Biotec cs columns
    Western blot analysis of type IX collagen CB-peptides from spondylolisthesis disc with Trp3 polymorphism (D9341). The collagen CB-peptides were partially resolved by reverse-phase <t>HPLC,</t> column fractions were run on <t>SDS-</t> 12.5% PAGE and then transblotted
    Cs Columns, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 99/100, based on 174 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad uno q1 column
    Western blot analysis of type IX collagen CB-peptides from spondylolisthesis disc with Trp3 polymorphism (D9341). The collagen CB-peptides were partially resolved by reverse-phase <t>HPLC,</t> column fractions were run on <t>SDS-</t> 12.5% PAGE and then transblotted
    Uno Q1 Column, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 114 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore petroleum ether
    Western blot analysis of type IX collagen CB-peptides from spondylolisthesis disc with Trp3 polymorphism (D9341). The collagen CB-peptides were partially resolved by reverse-phase <t>HPLC,</t> column fractions were run on <t>SDS-</t> 12.5% PAGE and then transblotted
    Petroleum Ether, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 194 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    GE Healthcare frac 950 fraction collector
    Western blot analysis of type IX collagen CB-peptides from spondylolisthesis disc with Trp3 polymorphism (D9341). The collagen CB-peptides were partially resolved by reverse-phase <t>HPLC,</t> column fractions were run on <t>SDS-</t> 12.5% PAGE and then transblotted
    Frac 950 Fraction Collector, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher nab protein a g spin columns
    Western blot analysis of type IX collagen CB-peptides from spondylolisthesis disc with Trp3 polymorphism (D9341). The collagen CB-peptides were partially resolved by reverse-phase <t>HPLC,</t> column fractions were run on <t>SDS-</t> 12.5% PAGE and then transblotted
    Nab Protein A G Spin Columns, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Miltenyi Biotec automacs separator column
    Western blot analysis of type IX collagen CB-peptides from spondylolisthesis disc with Trp3 polymorphism (D9341). The collagen CB-peptides were partially resolved by reverse-phase <t>HPLC,</t> column fractions were run on <t>SDS-</t> 12.5% PAGE and then transblotted
    Automacs Separator Column, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 99/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    GE Healthcare hitrapq hp column
    Western blot analysis of type IX collagen CB-peptides from spondylolisthesis disc with Trp3 polymorphism (D9341). The collagen CB-peptides were partially resolved by reverse-phase <t>HPLC,</t> column fractions were run on <t>SDS-</t> 12.5% PAGE and then transblotted
    Hitrapq Hp Column, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 96/100, based on 200 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dionex ionpac as24 analytical guard columns
    Western blot analysis of type IX collagen CB-peptides from spondylolisthesis disc with Trp3 polymorphism (D9341). The collagen CB-peptides were partially resolved by reverse-phase <t>HPLC,</t> column fractions were run on <t>SDS-</t> 12.5% PAGE and then transblotted
    Dionex Ionpac As24 Analytical Guard Columns, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad econo column bio rad column
    Western blot analysis of type IX collagen CB-peptides from spondylolisthesis disc with Trp3 polymorphism (D9341). The collagen CB-peptides were partially resolved by reverse-phase <t>HPLC,</t> column fractions were run on <t>SDS-</t> 12.5% PAGE and then transblotted
    Econo Column Bio Rad Column, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare superose 6 column
    PLK1 interacts with CtIP in a S327 phosphorylation-dependent manner. ( A ) Left: Purification scheme. 293T cells were pre-treated with nocodazole for 16 h and lysed. Total cell lysate (TCL) was applied to a pre-equilibrated Q-sepharose FF column. After washing, the bound proteins were eluted with 300 mM NaCl, followed by fractionation on a <t>Superose</t> 6 gel filtration column. Right: Western blotting was performed using the indicated antibodies on alternate fractions to monitor the elution of CtIP and PLK1. Superose 6 fractions (10–13, indicated by the up-arrow) contained both CtIP and PLK1. ( B ) 293T cell extracts were subjected to immunoprecipitation followed by western blotting with the indicated antibodies. ( C ) Unperturbed and nocodazole-arrested 293T cells, pre-treated with or without the indicated drugs, were lysed and subjected to immunoprecipitation followed by western blotting with the indicated antibodies. ( D ) Top: Twelve putative CDK consensus sites (SP/TP motifs) on CtIP. Bottom: the indicated CtIP constructs were expressed in Sf9 insect cells by baculovirus infection and then used in GST pull-down assays using purified GST-PBD protein as bait. Western blotting was performed to analyze the binding between PBD and CtIP variants. ( E ) U2OS cells were treated with nocodazole (NOC) for 16 h and then subjected to immunoprecipitation followed by western blotting with the indicated antibodies. ( F ) 293T cells were co-transfected with Myc-tagged CtIP variants and Flag-tagged PLK1 expression constructs. Cell extracts were subjected to immunoprecipitation followed by western blotting with the indicated antibodies. ( G ) The role of S327 phosphorylation in mediating the CtIP binding with PBD and subsequent phosphorylation of CtIP by PLK1. FL, flow through; KD, kinase domain.
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    Summary of cDNAs recovered from DENV-resistant Huh7.5 cell clones. Fifty-two colonies of clonal Huh7.5 cells that survived a chellenge infection of DENV-2 were infected with DENV at an MOI of 1, and culture supernatants of infected cells were subjected to plaque assay. mRNA were isolated from cells whose supernatant formed 10 or less DENV plaques less (DENV-resistant clones, total 32 clones), and cDNA inserts expressing in the clones were analyzed by sequencing. More than 50 plaques were formed with a supernatant of control protein (BAP)-expressing Huh7.5 cells.

    Journal: PLoS Pathogens

    Article Title: Characterization of RyDEN (C19orf66) as an Interferon-Stimulated Cellular Inhibitor against Dengue Virus Replication

    doi: 10.1371/journal.ppat.1005357

    Figure Lengend Snippet: Summary of cDNAs recovered from DENV-resistant Huh7.5 cell clones. Fifty-two colonies of clonal Huh7.5 cells that survived a chellenge infection of DENV-2 were infected with DENV at an MOI of 1, and culture supernatants of infected cells were subjected to plaque assay. mRNA were isolated from cells whose supernatant formed 10 or less DENV plaques less (DENV-resistant clones, total 32 clones), and cDNA inserts expressing in the clones were analyzed by sequencing. More than 50 plaques were formed with a supernatant of control protein (BAP)-expressing Huh7.5 cells.

    Article Snippet: The cDNA was synthesized using the CloneMiner cDNA Library Construction Kit (Life Technologies) from 3 μg of mRNA and fractionated with cDNA Size Fractionation Columns (Life Technologies).

    Techniques: Clone Assay, Infection, Plaque Assay, Isolation, Expressing, Sequencing

    Identification of RyDEN. (A) Procedure for gain-of-function screen. The cDNA library was generated from mRNA of IFN-α/ω-treated HeLa cells and transferred into a lentiviral vector by the Gateway recombination system. Infectious lentiviral vectors carrying the IFN cDNA library were produced as a VSV-G-pseudotyped virus and used to transduce DENV-susceptible Huh7.5 cells. cDNA library-expressing Huh7.5 cells were then challenged with DENV-2 at an MOI of 1, and cell colonies that survived DENV-induced cell death were collected. (B) Histogram analysis of cDNA fragments in library vectors. The entry vector (pDONR221, left panel) and destination vector (pYK005C, right panel) recombinated with the Gateway-compatible cDNA library were applied to Escherichia coli ( E . coli) , and the cells were spread onto LB plates to develop bacterial colonies. The cDNA fragments in individual colonies were amplified by PCR using primers described in Materials and Methods and visualized with agarose gel electrophoresis. The size of the PCR fragment was estimated by comparing the migration distance of the DNA molecular weight markers. Up to sixty colonies were picked up from each vector-transformed E . coli plate and analyzed. (C) Validation of DENV-resistant cell clones. Surviving clones obtained from (A) were seeded in a chamber slide and infected with DENV-2 at an MOI of 5. Two days after infection, cells were fixed with paraformaldehyde, permeabilized, and stained with anti-dsRNA antibody, followed by detection with Alexa Fluor 488-conjugated secondary antibody (red). Cell nuclei were stained with DAPI (blue). Representative merged images using four surviving clones (#1, 13, 14, and 15) and control cells (bacterial alkaline phosphatase [BAP]-expressing Huh7.5 cells) are shown. In a parallel experiment, the culture supernatant of infected cells was harvested 2 days after infection and subjected to plaque assay to measure the virus titer (insets). (D) Amplification of cDNA from DENV-resistant cells. Genomic DNA was isolated from cell clones, whose resistant property had been confirmed in (B), and cDNA was amplified by PCR using primers specific to the lentiviral vector. PCR products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. (E) Amino acid sequence of RyDEN. (F) Predicted domain organization of RyDEN. RyDEN protein (291 amino acid) was suggested to contain eight α-helixes (blue), seven β-strands (orange), NLS (121–137), NES (261–269), zinc-ribbon domain (112–135), and coiled-coil motif (261–285). A unique glutamic acid-rich (E-rich) domain was also found in the C-terminus.

    Journal: PLoS Pathogens

    Article Title: Characterization of RyDEN (C19orf66) as an Interferon-Stimulated Cellular Inhibitor against Dengue Virus Replication

    doi: 10.1371/journal.ppat.1005357

    Figure Lengend Snippet: Identification of RyDEN. (A) Procedure for gain-of-function screen. The cDNA library was generated from mRNA of IFN-α/ω-treated HeLa cells and transferred into a lentiviral vector by the Gateway recombination system. Infectious lentiviral vectors carrying the IFN cDNA library were produced as a VSV-G-pseudotyped virus and used to transduce DENV-susceptible Huh7.5 cells. cDNA library-expressing Huh7.5 cells were then challenged with DENV-2 at an MOI of 1, and cell colonies that survived DENV-induced cell death were collected. (B) Histogram analysis of cDNA fragments in library vectors. The entry vector (pDONR221, left panel) and destination vector (pYK005C, right panel) recombinated with the Gateway-compatible cDNA library were applied to Escherichia coli ( E . coli) , and the cells were spread onto LB plates to develop bacterial colonies. The cDNA fragments in individual colonies were amplified by PCR using primers described in Materials and Methods and visualized with agarose gel electrophoresis. The size of the PCR fragment was estimated by comparing the migration distance of the DNA molecular weight markers. Up to sixty colonies were picked up from each vector-transformed E . coli plate and analyzed. (C) Validation of DENV-resistant cell clones. Surviving clones obtained from (A) were seeded in a chamber slide and infected with DENV-2 at an MOI of 5. Two days after infection, cells were fixed with paraformaldehyde, permeabilized, and stained with anti-dsRNA antibody, followed by detection with Alexa Fluor 488-conjugated secondary antibody (red). Cell nuclei were stained with DAPI (blue). Representative merged images using four surviving clones (#1, 13, 14, and 15) and control cells (bacterial alkaline phosphatase [BAP]-expressing Huh7.5 cells) are shown. In a parallel experiment, the culture supernatant of infected cells was harvested 2 days after infection and subjected to plaque assay to measure the virus titer (insets). (D) Amplification of cDNA from DENV-resistant cells. Genomic DNA was isolated from cell clones, whose resistant property had been confirmed in (B), and cDNA was amplified by PCR using primers specific to the lentiviral vector. PCR products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. (E) Amino acid sequence of RyDEN. (F) Predicted domain organization of RyDEN. RyDEN protein (291 amino acid) was suggested to contain eight α-helixes (blue), seven β-strands (orange), NLS (121–137), NES (261–269), zinc-ribbon domain (112–135), and coiled-coil motif (261–285). A unique glutamic acid-rich (E-rich) domain was also found in the C-terminus.

    Article Snippet: The cDNA was synthesized using the CloneMiner cDNA Library Construction Kit (Life Technologies) from 3 μg of mRNA and fractionated with cDNA Size Fractionation Columns (Life Technologies).

    Techniques: cDNA Library Assay, Generated, Plasmid Preparation, Produced, Transduction, Expressing, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Migration, Molecular Weight, Transformation Assay, Clone Assay, Infection, Staining, Plaque Assay, Isolation, Sequencing

    Application of the new method to the construction of newborn mouse kidney DNA libraries. ( A ) Size distributions of amplified cDNAs were analyzed on 0.7% agarose gels. Lane 1 , DNA size marker. Lane 2 , previous method. The cDNAs were tagged by LL-Sal3, and amplified by the PCR condition VI with 1 μL of LL-Sal3A primer. Lane 3 , new method. The cDNAs were tagged with LL-Sal4 and amplified by the PCR condition III with 1 μL of primer S. Lane 4 , new method. The cDNAs were tagged with LL-Sal4 and amplified by the PCR condition V with 1 μL of primer L. ( B ) Comparison of size distributions of cDNA inserts between the short and long cDNA libraries. ( C ) Analysis of the complexity of cDNA libraries based on NCBI Mouse UniGene Index. ( D ) Sequence analyses of the long- and short-insert cDNA libraries. “Correct 3′-end” means that the 3′ end of cDNA sequence shows exact match to the 3′-end sequence of GenBank entry. “Others” means that the 3′ end of cDNA sequences does not match to the 3′ end sequence of GenBank entry. This can happen when either correct 3′-end sequences are not reported in the GenBank or our cDNA clones are truncated at 3′ ends. Although most cDNA clones have polyA sequence at the 3′ end, possible 3′-end truncation by Oligo(dT) mispriming cannot be excluded.

    Journal: Genome Research

    Article Title: Construction of Long-Transcript Enriched cDNA Libraries from Submicrogram Amounts of Total RNAs by a Universal PCR Amplification Method

    doi: 10.1101/gr.185501

    Figure Lengend Snippet: Application of the new method to the construction of newborn mouse kidney DNA libraries. ( A ) Size distributions of amplified cDNAs were analyzed on 0.7% agarose gels. Lane 1 , DNA size marker. Lane 2 , previous method. The cDNAs were tagged by LL-Sal3, and amplified by the PCR condition VI with 1 μL of LL-Sal3A primer. Lane 3 , new method. The cDNAs were tagged with LL-Sal4 and amplified by the PCR condition III with 1 μL of primer S. Lane 4 , new method. The cDNAs were tagged with LL-Sal4 and amplified by the PCR condition V with 1 μL of primer L. ( B ) Comparison of size distributions of cDNA inserts between the short and long cDNA libraries. ( C ) Analysis of the complexity of cDNA libraries based on NCBI Mouse UniGene Index. ( D ) Sequence analyses of the long- and short-insert cDNA libraries. “Correct 3′-end” means that the 3′ end of cDNA sequence shows exact match to the 3′-end sequence of GenBank entry. “Others” means that the 3′ end of cDNA sequences does not match to the 3′ end sequence of GenBank entry. This can happen when either correct 3′-end sequences are not reported in the GenBank or our cDNA clones are truncated at 3′ ends. Although most cDNA clones have polyA sequence at the 3′ end, possible 3′-end truncation by Oligo(dT) mispriming cannot be excluded.

    Article Snippet: The cDNAs were separated from the linkers by a cDNA size fractionation column (Life Technologies) and subjected to the ethanol precipitation.

    Techniques: Amplification, Marker, Polymerase Chain Reaction, Sequencing, Clone Assay

    IL4 gene therapy induces M2 markers and IL6 expression in CNS-infiltrating CD11b + cells in vivo. mRNA levels of IL6, IL-1β, and YM1 were measured by real-time RT-PCR in CD11b + cells recovered from the CNS of EAE mice treated intracisternally with an IL4-expressing (IL4) or a GFP-expressing (GFP) lentivirus. IL4 gene therapy induces a decrease of IL-1β ( a ), and an increase of IL6 ( b ) and Ym1 ( c ), as compared to control-treated mice. n = 3 for each group. Gapdh has been used as a housekeeping gene. Data are shown as arbitrary units (AU ± standard deviation). * P

    Journal: Journal of Neuroinflammation

    Article Title: IL4 induces IL6-producing M2 macrophages associated to inhibition of neuroinflammation in vitro and in vivo

    doi: 10.1186/s12974-016-0596-5

    Figure Lengend Snippet: IL4 gene therapy induces M2 markers and IL6 expression in CNS-infiltrating CD11b + cells in vivo. mRNA levels of IL6, IL-1β, and YM1 were measured by real-time RT-PCR in CD11b + cells recovered from the CNS of EAE mice treated intracisternally with an IL4-expressing (IL4) or a GFP-expressing (GFP) lentivirus. IL4 gene therapy induces a decrease of IL-1β ( a ), and an increase of IL6 ( b ) and Ym1 ( c ), as compared to control-treated mice. n = 3 for each group. Gapdh has been used as a housekeeping gene. Data are shown as arbitrary units (AU ± standard deviation). * P

    Article Snippet: Finally, the cells were loaded on the MS-columns and the column-bound CD11b+ fraction isolated (Myltenyi Biotech).

    Techniques: Expressing, In Vivo, Quantitative RT-PCR, Mouse Assay, Standard Deviation

    Western blot analysis of type IX collagen CB-peptides from spondylolisthesis disc with Trp3 polymorphism (D9341). The collagen CB-peptides were partially resolved by reverse-phase HPLC, column fractions were run on SDS- 12.5% PAGE and then transblotted

    Journal: Spine

    Article Title: Type IX Collagen Neo-Deposition in Degenerative Discs of Surgical Patients Whether Genotyped Plus or Minus for COL9 Risk Alleles

    doi: 10.1097/BRS.0b013e3181ffdd61

    Figure Lengend Snippet: Western blot analysis of type IX collagen CB-peptides from spondylolisthesis disc with Trp3 polymorphism (D9341). The collagen CB-peptides were partially resolved by reverse-phase HPLC, column fractions were run on SDS- 12.5% PAGE and then transblotted

    Article Snippet: Reverse-phase HPLC fractions of CNBr-peptides were run on SDS-12.5% (w/w) polyacrylamide gels and transblotted to a nitrocellulose membrane (Bio-Rad Laboratories) using a Milliblot-SDE electroblotting apparatus (Millipore).

    Techniques: Western Blot, High Performance Liquid Chromatography, Polyacrylamide Gel Electrophoresis

    PLK1 interacts with CtIP in a S327 phosphorylation-dependent manner. ( A ) Left: Purification scheme. 293T cells were pre-treated with nocodazole for 16 h and lysed. Total cell lysate (TCL) was applied to a pre-equilibrated Q-sepharose FF column. After washing, the bound proteins were eluted with 300 mM NaCl, followed by fractionation on a Superose 6 gel filtration column. Right: Western blotting was performed using the indicated antibodies on alternate fractions to monitor the elution of CtIP and PLK1. Superose 6 fractions (10–13, indicated by the up-arrow) contained both CtIP and PLK1. ( B ) 293T cell extracts were subjected to immunoprecipitation followed by western blotting with the indicated antibodies. ( C ) Unperturbed and nocodazole-arrested 293T cells, pre-treated with or without the indicated drugs, were lysed and subjected to immunoprecipitation followed by western blotting with the indicated antibodies. ( D ) Top: Twelve putative CDK consensus sites (SP/TP motifs) on CtIP. Bottom: the indicated CtIP constructs were expressed in Sf9 insect cells by baculovirus infection and then used in GST pull-down assays using purified GST-PBD protein as bait. Western blotting was performed to analyze the binding between PBD and CtIP variants. ( E ) U2OS cells were treated with nocodazole (NOC) for 16 h and then subjected to immunoprecipitation followed by western blotting with the indicated antibodies. ( F ) 293T cells were co-transfected with Myc-tagged CtIP variants and Flag-tagged PLK1 expression constructs. Cell extracts were subjected to immunoprecipitation followed by western blotting with the indicated antibodies. ( G ) The role of S327 phosphorylation in mediating the CtIP binding with PBD and subsequent phosphorylation of CtIP by PLK1. FL, flow through; KD, kinase domain.

    Journal: Nucleic Acids Research

    Article Title: PLK1 targets CtIP to promote microhomology-mediated end joining

    doi: 10.1093/nar/gky810

    Figure Lengend Snippet: PLK1 interacts with CtIP in a S327 phosphorylation-dependent manner. ( A ) Left: Purification scheme. 293T cells were pre-treated with nocodazole for 16 h and lysed. Total cell lysate (TCL) was applied to a pre-equilibrated Q-sepharose FF column. After washing, the bound proteins were eluted with 300 mM NaCl, followed by fractionation on a Superose 6 gel filtration column. Right: Western blotting was performed using the indicated antibodies on alternate fractions to monitor the elution of CtIP and PLK1. Superose 6 fractions (10–13, indicated by the up-arrow) contained both CtIP and PLK1. ( B ) 293T cell extracts were subjected to immunoprecipitation followed by western blotting with the indicated antibodies. ( C ) Unperturbed and nocodazole-arrested 293T cells, pre-treated with or without the indicated drugs, were lysed and subjected to immunoprecipitation followed by western blotting with the indicated antibodies. ( D ) Top: Twelve putative CDK consensus sites (SP/TP motifs) on CtIP. Bottom: the indicated CtIP constructs were expressed in Sf9 insect cells by baculovirus infection and then used in GST pull-down assays using purified GST-PBD protein as bait. Western blotting was performed to analyze the binding between PBD and CtIP variants. ( E ) U2OS cells were treated with nocodazole (NOC) for 16 h and then subjected to immunoprecipitation followed by western blotting with the indicated antibodies. ( F ) 293T cells were co-transfected with Myc-tagged CtIP variants and Flag-tagged PLK1 expression constructs. Cell extracts were subjected to immunoprecipitation followed by western blotting with the indicated antibodies. ( G ) The role of S327 phosphorylation in mediating the CtIP binding with PBD and subsequent phosphorylation of CtIP by PLK1. FL, flow through; KD, kinase domain.

    Article Snippet: The 300 mM fraction was then added to a Superose 6 column (GE Healthcare).

    Techniques: Purification, Fractionation, Filtration, Western Blot, Immunoprecipitation, Construct, Infection, Binding Assay, Transfection, Expressing, Flow Cytometry

    The conserved C-terminal region of Bud13 is sufficient to mediate interaction with Snu17 and shows similarity with UHM ligand motifs (ULM). ( A ) Schematic representation of yeast Bud13 showing the location of the most conserved region (amino acids 201–266) as a black rectangle. An alignment of part of the sequence of this region with the sequence of the human homologue (hBud13/MGC13125) is presented below. Underneath this alignment, the sequence of the ULM of the SF1 splicing factor, which as been shown to be sufficient to bind to the third RRM of U2AF 65 ( 35 ), is presented to demonstrate sequence similarity. ( B ) A recombinant peptide encompassing residues 201–266 of Bud13 was mixed with recombinant 6His-Snu17 (Input, lane 1) and the two factors were shown to interact by co-fractionation on a Superdex 75 column (GE Healthcare) (see Elution fractions, lanes 2–10). Control fractionation demonstrated that the Bud13 peptide which contains its first 201 residues elutes later under the same conditions. ( C ) SDS–PAGE of samples corresponding to the complex between Snu17 and the Bud13 fragment 201–266. The sample which had been injected onto the gel-filtration column was migrated on lane 1, while lanes 2–10 correspond to the peak fractions indicated beneath the chromatograms in B). Standard molecular weight markers were migrated alongside for comparison (lane 11). Lane 2 most likely contains carry-over from the input protein.

    Journal: Nucleic Acids Research

    Article Title: Structure of the yeast Pml1 splicing factor and its integration into the RES complex

    doi: 10.1093/nar/gkn894

    Figure Lengend Snippet: The conserved C-terminal region of Bud13 is sufficient to mediate interaction with Snu17 and shows similarity with UHM ligand motifs (ULM). ( A ) Schematic representation of yeast Bud13 showing the location of the most conserved region (amino acids 201–266) as a black rectangle. An alignment of part of the sequence of this region with the sequence of the human homologue (hBud13/MGC13125) is presented below. Underneath this alignment, the sequence of the ULM of the SF1 splicing factor, which as been shown to be sufficient to bind to the third RRM of U2AF 65 ( 35 ), is presented to demonstrate sequence similarity. ( B ) A recombinant peptide encompassing residues 201–266 of Bud13 was mixed with recombinant 6His-Snu17 (Input, lane 1) and the two factors were shown to interact by co-fractionation on a Superdex 75 column (GE Healthcare) (see Elution fractions, lanes 2–10). Control fractionation demonstrated that the Bud13 peptide which contains its first 201 residues elutes later under the same conditions. ( C ) SDS–PAGE of samples corresponding to the complex between Snu17 and the Bud13 fragment 201–266. The sample which had been injected onto the gel-filtration column was migrated on lane 1, while lanes 2–10 correspond to the peak fractions indicated beneath the chromatograms in B). Standard molecular weight markers were migrated alongside for comparison (lane 11). Lane 2 most likely contains carry-over from the input protein.

    Article Snippet: The peak fractions were purified on a Superdex 75 column (GE Healthcare, Buckinghamshire, UK), and concentrated using Vivaspin 5000 nominal molecular weight limit cut-off centrifugal concentrators (Vivascience, Goettingen, Germany).

    Techniques: Sequencing, Recombinant, Fractionation, SDS Page, Injection, Filtration, Molecular Weight