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  • 99
    Thermo Fisher rnaseout ribonuclease inhibitor
    Rnaseout Ribonuclease Inhibitor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 412 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher rnaseout
    SAMHD1 interacts with LINE-1. a 293T cells were transfected with the L1 expression vector pAD2TE1 together with plasmids encoding wt SAMHD1, SAMHD1 T592A, or SAMHD1 T592D. Two days posttransfection, cells were lysed and ORF1p-T7 was precipitated using anti-T7 antibody coupled to magnetic beads. The precipitates were analyzed by immunoblot. SAMHD1-myc, T592A-myc, and T592D-myc signals were quantified using AIDA Image Analyzer software and normalized on the T7 signal in IP (bait) and the myc signal in WCL (input). b 293T cells were transfected with expression plasmids for L1 ORF1p-FLAG or ORF2p-3xFLAG together with empty vector (pcDNA) or SAMHD1 T592A. Two days posttransfection, cells were lysed and L1 proteins were precipitated with anti-FLAG antibody coupled to magnetic beads. A representative quantification of the precipitated SAMHD1 T592A signal is shown. HRP signals were quantified with AIDA Image analyzer software and normalized on bait signal and WCL input signal. c 293T cells were transfected with a L1 expression plasmid together with empty vector (pcDNA), SAMHD1 T592A, or MOV10. Cells were lysed 2 days posttransfection. Lysates were treated with either <t>RNaseOUT</t> or 15 μg/ml RNaseA prior to ORF1p-T7 precipitation using anti-T7 antibody coupled to magnetic beads. d 293T cells were transfected with empty vector (pcDNA), L1 expression vector and expression plasmids for SAMHD1 wt, T592A, or T592D. Cells were lysed 2 days posttransfection and SAMHD1 was precipitated with an anti-myc antibody coupled to magnetic beads. Subsequently, MLV-RT and LEAP-RT reactions were performed and amplification products were visualized on a 2% agarose gel. Input protein content was controlled by immunoblot. One out of three independent experiments is shown. WCL: Whole cell lysate, IP: immunoprecipitation
    Rnaseout, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 11523 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher ¼l rnaseout
    SAMHD1 interacts with LINE-1. a 293T cells were transfected with the L1 expression vector pAD2TE1 together with plasmids encoding wt SAMHD1, SAMHD1 T592A, or SAMHD1 T592D. Two days posttransfection, cells were lysed and ORF1p-T7 was precipitated using anti-T7 antibody coupled to magnetic beads. The precipitates were analyzed by immunoblot. SAMHD1-myc, T592A-myc, and T592D-myc signals were quantified using AIDA Image Analyzer software and normalized on the T7 signal in IP (bait) and the myc signal in WCL (input). b 293T cells were transfected with expression plasmids for L1 ORF1p-FLAG or ORF2p-3xFLAG together with empty vector (pcDNA) or SAMHD1 T592A. Two days posttransfection, cells were lysed and L1 proteins were precipitated with anti-FLAG antibody coupled to magnetic beads. A representative quantification of the precipitated SAMHD1 T592A signal is shown. HRP signals were quantified with AIDA Image analyzer software and normalized on bait signal and WCL input signal. c 293T cells were transfected with a L1 expression plasmid together with empty vector (pcDNA), SAMHD1 T592A, or MOV10. Cells were lysed 2 days posttransfection. Lysates were treated with either <t>RNaseOUT</t> or 15 μg/ml RNaseA prior to ORF1p-T7 precipitation using anti-T7 antibody coupled to magnetic beads. d 293T cells were transfected with empty vector (pcDNA), L1 expression vector and expression plasmids for SAMHD1 wt, T592A, or T592D. Cells were lysed 2 days posttransfection and SAMHD1 was precipitated with an anti-myc antibody coupled to magnetic beads. Subsequently, MLV-RT and LEAP-RT reactions were performed and amplification products were visualized on a 2% agarose gel. Input protein content was controlled by immunoblot. One out of three independent experiments is shown. WCL: Whole cell lysate, IP: immunoprecipitation
    ¼l Rnaseout, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher trace rnaseout
    SAMHD1 interacts with LINE-1. a 293T cells were transfected with the L1 expression vector pAD2TE1 together with plasmids encoding wt SAMHD1, SAMHD1 T592A, or SAMHD1 T592D. Two days posttransfection, cells were lysed and ORF1p-T7 was precipitated using anti-T7 antibody coupled to magnetic beads. The precipitates were analyzed by immunoblot. SAMHD1-myc, T592A-myc, and T592D-myc signals were quantified using AIDA Image Analyzer software and normalized on the T7 signal in IP (bait) and the myc signal in WCL (input). b 293T cells were transfected with expression plasmids for L1 ORF1p-FLAG or ORF2p-3xFLAG together with empty vector (pcDNA) or SAMHD1 T592A. Two days posttransfection, cells were lysed and L1 proteins were precipitated with anti-FLAG antibody coupled to magnetic beads. A representative quantification of the precipitated SAMHD1 T592A signal is shown. HRP signals were quantified with AIDA Image analyzer software and normalized on bait signal and WCL input signal. c 293T cells were transfected with a L1 expression plasmid together with empty vector (pcDNA), SAMHD1 T592A, or MOV10. Cells were lysed 2 days posttransfection. Lysates were treated with either <t>RNaseOUT</t> or 15 μg/ml RNaseA prior to ORF1p-T7 precipitation using anti-T7 antibody coupled to magnetic beads. d 293T cells were transfected with empty vector (pcDNA), L1 expression vector and expression plasmids for SAMHD1 wt, T592A, or T592D. Cells were lysed 2 days posttransfection and SAMHD1 was precipitated with an anti-myc antibody coupled to magnetic beads. Subsequently, MLV-RT and LEAP-RT reactions were performed and amplification products were visualized on a 2% agarose gel. Input protein content was controlled by immunoblot. One out of three independent experiments is shown. WCL: Whole cell lysate, IP: immunoprecipitation
    Trace Rnaseout, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher un rnaseout
    SAMHD1 interacts with LINE-1. a 293T cells were transfected with the L1 expression vector pAD2TE1 together with plasmids encoding wt SAMHD1, SAMHD1 T592A, or SAMHD1 T592D. Two days posttransfection, cells were lysed and ORF1p-T7 was precipitated using anti-T7 antibody coupled to magnetic beads. The precipitates were analyzed by immunoblot. SAMHD1-myc, T592A-myc, and T592D-myc signals were quantified using AIDA Image Analyzer software and normalized on the T7 signal in IP (bait) and the myc signal in WCL (input). b 293T cells were transfected with expression plasmids for L1 ORF1p-FLAG or ORF2p-3xFLAG together with empty vector (pcDNA) or SAMHD1 T592A. Two days posttransfection, cells were lysed and L1 proteins were precipitated with anti-FLAG antibody coupled to magnetic beads. A representative quantification of the precipitated SAMHD1 T592A signal is shown. HRP signals were quantified with AIDA Image analyzer software and normalized on bait signal and WCL input signal. c 293T cells were transfected with a L1 expression plasmid together with empty vector (pcDNA), SAMHD1 T592A, or MOV10. Cells were lysed 2 days posttransfection. Lysates were treated with either <t>RNaseOUT</t> or 15 μg/ml RNaseA prior to ORF1p-T7 precipitation using anti-T7 antibody coupled to magnetic beads. d 293T cells were transfected with empty vector (pcDNA), L1 expression vector and expression plasmids for SAMHD1 wt, T592A, or T592D. Cells were lysed 2 days posttransfection and SAMHD1 was precipitated with an anti-myc antibody coupled to magnetic beads. Subsequently, MLV-RT and LEAP-RT reactions were performed and amplification products were visualized on a 2% agarose gel. Input protein content was controlled by immunoblot. One out of three independent experiments is shown. WCL: Whole cell lysate, IP: immunoprecipitation
    Un Rnaseout, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher rnasaout
    SAMHD1 interacts with LINE-1. a 293T cells were transfected with the L1 expression vector pAD2TE1 together with plasmids encoding wt SAMHD1, SAMHD1 T592A, or SAMHD1 T592D. Two days posttransfection, cells were lysed and ORF1p-T7 was precipitated using anti-T7 antibody coupled to magnetic beads. The precipitates were analyzed by immunoblot. SAMHD1-myc, T592A-myc, and T592D-myc signals were quantified using AIDA Image Analyzer software and normalized on the T7 signal in IP (bait) and the myc signal in WCL (input). b 293T cells were transfected with expression plasmids for L1 ORF1p-FLAG or ORF2p-3xFLAG together with empty vector (pcDNA) or SAMHD1 T592A. Two days posttransfection, cells were lysed and L1 proteins were precipitated with anti-FLAG antibody coupled to magnetic beads. A representative quantification of the precipitated SAMHD1 T592A signal is shown. HRP signals were quantified with AIDA Image analyzer software and normalized on bait signal and WCL input signal. c 293T cells were transfected with a L1 expression plasmid together with empty vector (pcDNA), SAMHD1 T592A, or MOV10. Cells were lysed 2 days posttransfection. Lysates were treated with either <t>RNaseOUT</t> or 15 μg/ml RNaseA prior to ORF1p-T7 precipitation using anti-T7 antibody coupled to magnetic beads. d 293T cells were transfected with empty vector (pcDNA), L1 expression vector and expression plasmids for SAMHD1 wt, T592A, or T592D. Cells were lysed 2 days posttransfection and SAMHD1 was precipitated with an anti-myc antibody coupled to magnetic beads. Subsequently, MLV-RT and LEAP-RT reactions were performed and amplification products were visualized on a 2% agarose gel. Input protein content was controlled by immunoblot. One out of three independent experiments is shown. WCL: Whole cell lysate, IP: immunoprecipitation
    Rnasaout, supplied by Thermo Fisher, 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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    99
    Thermo Fisher ribonuclease inhibitor
    SAMHD1 interacts with LINE-1. a 293T cells were transfected with the L1 expression vector pAD2TE1 together with plasmids encoding wt SAMHD1, SAMHD1 T592A, or SAMHD1 T592D. Two days posttransfection, cells were lysed and ORF1p-T7 was precipitated using anti-T7 antibody coupled to magnetic beads. The precipitates were analyzed by immunoblot. SAMHD1-myc, T592A-myc, and T592D-myc signals were quantified using AIDA Image Analyzer software and normalized on the T7 signal in IP (bait) and the myc signal in WCL (input). b 293T cells were transfected with expression plasmids for L1 ORF1p-FLAG or ORF2p-3xFLAG together with empty vector (pcDNA) or SAMHD1 T592A. Two days posttransfection, cells were lysed and L1 proteins were precipitated with anti-FLAG antibody coupled to magnetic beads. A representative quantification of the precipitated SAMHD1 T592A signal is shown. HRP signals were quantified with AIDA Image analyzer software and normalized on bait signal and WCL input signal. c 293T cells were transfected with a L1 expression plasmid together with empty vector (pcDNA), SAMHD1 T592A, or MOV10. Cells were lysed 2 days posttransfection. Lysates were treated with either <t>RNaseOUT</t> or 15 μg/ml RNaseA prior to ORF1p-T7 precipitation using anti-T7 antibody coupled to magnetic beads. d 293T cells were transfected with empty vector (pcDNA), L1 expression vector and expression plasmids for SAMHD1 wt, T592A, or T592D. Cells were lysed 2 days posttransfection and SAMHD1 was precipitated with an anti-myc antibody coupled to magnetic beads. Subsequently, MLV-RT and LEAP-RT reactions were performed and amplification products were visualized on a 2% agarose gel. Input protein content was controlled by immunoblot. One out of three independent experiments is shown. WCL: Whole cell lysate, IP: immunoprecipitation
    Ribonuclease Inhibitor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1142 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher rnasout
    SAMHD1 interacts with LINE-1. a 293T cells were transfected with the L1 expression vector pAD2TE1 together with plasmids encoding wt SAMHD1, SAMHD1 T592A, or SAMHD1 T592D. Two days posttransfection, cells were lysed and ORF1p-T7 was precipitated using anti-T7 antibody coupled to magnetic beads. The precipitates were analyzed by immunoblot. SAMHD1-myc, T592A-myc, and T592D-myc signals were quantified using AIDA Image Analyzer software and normalized on the T7 signal in IP (bait) and the myc signal in WCL (input). b 293T cells were transfected with expression plasmids for L1 ORF1p-FLAG or ORF2p-3xFLAG together with empty vector (pcDNA) or SAMHD1 T592A. Two days posttransfection, cells were lysed and L1 proteins were precipitated with anti-FLAG antibody coupled to magnetic beads. A representative quantification of the precipitated SAMHD1 T592A signal is shown. HRP signals were quantified with AIDA Image analyzer software and normalized on bait signal and WCL input signal. c 293T cells were transfected with a L1 expression plasmid together with empty vector (pcDNA), SAMHD1 T592A, or MOV10. Cells were lysed 2 days posttransfection. Lysates were treated with either <t>RNaseOUT</t> or 15 μg/ml RNaseA prior to ORF1p-T7 precipitation using anti-T7 antibody coupled to magnetic beads. d 293T cells were transfected with empty vector (pcDNA), L1 expression vector and expression plasmids for SAMHD1 wt, T592A, or T592D. Cells were lysed 2 days posttransfection and SAMHD1 was precipitated with an anti-myc antibody coupled to magnetic beads. Subsequently, MLV-RT and LEAP-RT reactions were performed and amplification products were visualized on a 2% agarose gel. Input protein content was controlled by immunoblot. One out of three independent experiments is shown. WCL: Whole cell lysate, IP: immunoprecipitation
    Rnasout, supplied by Thermo Fisher, 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 superscript iii rnaseout enzyme mix
    SAMHD1 interacts with LINE-1. a 293T cells were transfected with the L1 expression vector pAD2TE1 together with plasmids encoding wt SAMHD1, SAMHD1 T592A, or SAMHD1 T592D. Two days posttransfection, cells were lysed and ORF1p-T7 was precipitated using anti-T7 antibody coupled to magnetic beads. The precipitates were analyzed by immunoblot. SAMHD1-myc, T592A-myc, and T592D-myc signals were quantified using AIDA Image Analyzer software and normalized on the T7 signal in IP (bait) and the myc signal in WCL (input). b 293T cells were transfected with expression plasmids for L1 ORF1p-FLAG or ORF2p-3xFLAG together with empty vector (pcDNA) or SAMHD1 T592A. Two days posttransfection, cells were lysed and L1 proteins were precipitated with anti-FLAG antibody coupled to magnetic beads. A representative quantification of the precipitated SAMHD1 T592A signal is shown. HRP signals were quantified with AIDA Image analyzer software and normalized on bait signal and WCL input signal. c 293T cells were transfected with a L1 expression plasmid together with empty vector (pcDNA), SAMHD1 T592A, or MOV10. Cells were lysed 2 days posttransfection. Lysates were treated with either <t>RNaseOUT</t> or 15 μg/ml RNaseA prior to ORF1p-T7 precipitation using anti-T7 antibody coupled to magnetic beads. d 293T cells were transfected with empty vector (pcDNA), L1 expression vector and expression plasmids for SAMHD1 wt, T592A, or T592D. Cells were lysed 2 days posttransfection and SAMHD1 was precipitated with an anti-myc antibody coupled to magnetic beads. Subsequently, MLV-RT and LEAP-RT reactions were performed and amplification products were visualized on a 2% agarose gel. Input protein content was controlled by immunoblot. One out of three independent experiments is shown. WCL: Whole cell lysate, IP: immunoprecipitation
    Superscript Iii Rnaseout Enzyme Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher 0 5ã‚â ãŽâ¼l rnaseout
    SAMHD1 interacts with LINE-1. a 293T cells were transfected with the L1 expression vector pAD2TE1 together with plasmids encoding wt SAMHD1, SAMHD1 T592A, or SAMHD1 T592D. Two days posttransfection, cells were lysed and ORF1p-T7 was precipitated using anti-T7 antibody coupled to magnetic beads. The precipitates were analyzed by immunoblot. SAMHD1-myc, T592A-myc, and T592D-myc signals were quantified using AIDA Image Analyzer software and normalized on the T7 signal in IP (bait) and the myc signal in WCL (input). b 293T cells were transfected with expression plasmids for L1 ORF1p-FLAG or ORF2p-3xFLAG together with empty vector (pcDNA) or SAMHD1 T592A. Two days posttransfection, cells were lysed and L1 proteins were precipitated with anti-FLAG antibody coupled to magnetic beads. A representative quantification of the precipitated SAMHD1 T592A signal is shown. HRP signals were quantified with AIDA Image analyzer software and normalized on bait signal and WCL input signal. c 293T cells were transfected with a L1 expression plasmid together with empty vector (pcDNA), SAMHD1 T592A, or MOV10. Cells were lysed 2 days posttransfection. Lysates were treated with either <t>RNaseOUT</t> or 15 μg/ml RNaseA prior to ORF1p-T7 precipitation using anti-T7 antibody coupled to magnetic beads. d 293T cells were transfected with empty vector (pcDNA), L1 expression vector and expression plasmids for SAMHD1 wt, T592A, or T592D. Cells were lysed 2 days posttransfection and SAMHD1 was precipitated with an anti-myc antibody coupled to magnetic beads. Subsequently, MLV-RT and LEAP-RT reactions were performed and amplification products were visualized on a 2% agarose gel. Input protein content was controlled by immunoblot. One out of three independent experiments is shown. WCL: Whole cell lysate, IP: immunoprecipitation
    0 5ã‚â ãŽâ¼l Rnaseout, supplied by Thermo Fisher, 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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    80
    Thermo Fisher rnaseoutã¢â„â¢
    SAMHD1 interacts with LINE-1. a 293T cells were transfected with the L1 expression vector pAD2TE1 together with plasmids encoding wt SAMHD1, SAMHD1 T592A, or SAMHD1 T592D. Two days posttransfection, cells were lysed and ORF1p-T7 was precipitated using anti-T7 antibody coupled to magnetic beads. The precipitates were analyzed by immunoblot. SAMHD1-myc, T592A-myc, and T592D-myc signals were quantified using AIDA Image Analyzer software and normalized on the T7 signal in IP (bait) and the myc signal in WCL (input). b 293T cells were transfected with expression plasmids for L1 ORF1p-FLAG or ORF2p-3xFLAG together with empty vector (pcDNA) or SAMHD1 T592A. Two days posttransfection, cells were lysed and L1 proteins were precipitated with anti-FLAG antibody coupled to magnetic beads. A representative quantification of the precipitated SAMHD1 T592A signal is shown. HRP signals were quantified with AIDA Image analyzer software and normalized on bait signal and WCL input signal. c 293T cells were transfected with a L1 expression plasmid together with empty vector (pcDNA), SAMHD1 T592A, or MOV10. Cells were lysed 2 days posttransfection. Lysates were treated with either <t>RNaseOUT</t> or 15 μg/ml RNaseA prior to ORF1p-T7 precipitation using anti-T7 antibody coupled to magnetic beads. d 293T cells were transfected with empty vector (pcDNA), L1 expression vector and expression plasmids for SAMHD1 wt, T592A, or T592D. Cells were lysed 2 days posttransfection and SAMHD1 was precipitated with an anti-myc antibody coupled to magnetic beads. Subsequently, MLV-RT and LEAP-RT reactions were performed and amplification products were visualized on a 2% agarose gel. Input protein content was controlled by immunoblot. One out of three independent experiments is shown. WCL: Whole cell lysate, IP: immunoprecipitation
    Rnaseoutã¢â„â¢, supplied by Thermo Fisher, 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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    86
    Thermo Fisher rnasa
    SAMHD1 interacts with LINE-1. a 293T cells were transfected with the L1 expression vector pAD2TE1 together with plasmids encoding wt SAMHD1, SAMHD1 T592A, or SAMHD1 T592D. Two days posttransfection, cells were lysed and ORF1p-T7 was precipitated using anti-T7 antibody coupled to magnetic beads. The precipitates were analyzed by immunoblot. SAMHD1-myc, T592A-myc, and T592D-myc signals were quantified using AIDA Image Analyzer software and normalized on the T7 signal in IP (bait) and the myc signal in WCL (input). b 293T cells were transfected with expression plasmids for L1 ORF1p-FLAG or ORF2p-3xFLAG together with empty vector (pcDNA) or SAMHD1 T592A. Two days posttransfection, cells were lysed and L1 proteins were precipitated with anti-FLAG antibody coupled to magnetic beads. A representative quantification of the precipitated SAMHD1 T592A signal is shown. HRP signals were quantified with AIDA Image analyzer software and normalized on bait signal and WCL input signal. c 293T cells were transfected with a L1 expression plasmid together with empty vector (pcDNA), SAMHD1 T592A, or MOV10. Cells were lysed 2 days posttransfection. Lysates were treated with either <t>RNaseOUT</t> or 15 μg/ml RNaseA prior to ORF1p-T7 precipitation using anti-T7 antibody coupled to magnetic beads. d 293T cells were transfected with empty vector (pcDNA), L1 expression vector and expression plasmids for SAMHD1 wt, T592A, or T592D. Cells were lysed 2 days posttransfection and SAMHD1 was precipitated with an anti-myc antibody coupled to magnetic beads. Subsequently, MLV-RT and LEAP-RT reactions were performed and amplification products were visualized on a 2% agarose gel. Input protein content was controlled by immunoblot. One out of three independent experiments is shown. WCL: Whole cell lysate, IP: immunoprecipitation
    Rnasa, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rnaseou
    SAMHD1 interacts with LINE-1. a 293T cells were transfected with the L1 expression vector pAD2TE1 together with plasmids encoding wt SAMHD1, SAMHD1 T592A, or SAMHD1 T592D. Two days posttransfection, cells were lysed and ORF1p-T7 was precipitated using anti-T7 antibody coupled to magnetic beads. The precipitates were analyzed by immunoblot. SAMHD1-myc, T592A-myc, and T592D-myc signals were quantified using AIDA Image Analyzer software and normalized on the T7 signal in IP (bait) and the myc signal in WCL (input). b 293T cells were transfected with expression plasmids for L1 ORF1p-FLAG or ORF2p-3xFLAG together with empty vector (pcDNA) or SAMHD1 T592A. Two days posttransfection, cells were lysed and L1 proteins were precipitated with anti-FLAG antibody coupled to magnetic beads. A representative quantification of the precipitated SAMHD1 T592A signal is shown. HRP signals were quantified with AIDA Image analyzer software and normalized on bait signal and WCL input signal. c 293T cells were transfected with a L1 expression plasmid together with empty vector (pcDNA), SAMHD1 T592A, or MOV10. Cells were lysed 2 days posttransfection. Lysates were treated with either <t>RNaseOUT</t> or 15 μg/ml RNaseA prior to ORF1p-T7 precipitation using anti-T7 antibody coupled to magnetic beads. d 293T cells were transfected with empty vector (pcDNA), L1 expression vector and expression plasmids for SAMHD1 wt, T592A, or T592D. Cells were lysed 2 days posttransfection and SAMHD1 was precipitated with an anti-myc antibody coupled to magnetic beads. Subsequently, MLV-RT and LEAP-RT reactions were performed and amplification products were visualized on a 2% agarose gel. Input protein content was controlled by immunoblot. One out of three independent experiments is shown. WCL: Whole cell lysate, IP: immunoprecipitation
    Rnaseou, supplied by Thermo Fisher, 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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    93
    Thermo Fisher 40 u rnaseout
    SAMHD1 interacts with LINE-1. a 293T cells were transfected with the L1 expression vector pAD2TE1 together with plasmids encoding wt SAMHD1, SAMHD1 T592A, or SAMHD1 T592D. Two days posttransfection, cells were lysed and ORF1p-T7 was precipitated using anti-T7 antibody coupled to magnetic beads. The precipitates were analyzed by immunoblot. SAMHD1-myc, T592A-myc, and T592D-myc signals were quantified using AIDA Image Analyzer software and normalized on the T7 signal in IP (bait) and the myc signal in WCL (input). b 293T cells were transfected with expression plasmids for L1 ORF1p-FLAG or ORF2p-3xFLAG together with empty vector (pcDNA) or SAMHD1 T592A. Two days posttransfection, cells were lysed and L1 proteins were precipitated with anti-FLAG antibody coupled to magnetic beads. A representative quantification of the precipitated SAMHD1 T592A signal is shown. HRP signals were quantified with AIDA Image analyzer software and normalized on bait signal and WCL input signal. c 293T cells were transfected with a L1 expression plasmid together with empty vector (pcDNA), SAMHD1 T592A, or MOV10. Cells were lysed 2 days posttransfection. Lysates were treated with either <t>RNaseOUT</t> or 15 μg/ml RNaseA prior to ORF1p-T7 precipitation using anti-T7 antibody coupled to magnetic beads. d 293T cells were transfected with empty vector (pcDNA), L1 expression vector and expression plasmids for SAMHD1 wt, T592A, or T592D. Cells were lysed 2 days posttransfection and SAMHD1 was precipitated with an anti-myc antibody coupled to magnetic beads. Subsequently, MLV-RT and LEAP-RT reactions were performed and amplification products were visualized on a 2% agarose gel. Input protein content was controlled by immunoblot. One out of three independent experiments is shown. WCL: Whole cell lysate, IP: immunoprecipitation
    40 U Rnaseout, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript iii reverse transcriptase ssiiirt
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    Superscript Iii Reverse Transcriptase Ssiiirt, supplied by Thermo Fisher, 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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    SAMHD1 interacts with LINE-1. a 293T cells were transfected with the L1 expression vector pAD2TE1 together with plasmids encoding wt SAMHD1, SAMHD1 T592A, or SAMHD1 T592D. Two days posttransfection, cells were lysed and ORF1p-T7 was precipitated using anti-T7 antibody coupled to magnetic beads. The precipitates were analyzed by immunoblot. SAMHD1-myc, T592A-myc, and T592D-myc signals were quantified using AIDA Image Analyzer software and normalized on the T7 signal in IP (bait) and the myc signal in WCL (input). b 293T cells were transfected with expression plasmids for L1 ORF1p-FLAG or ORF2p-3xFLAG together with empty vector (pcDNA) or SAMHD1 T592A. Two days posttransfection, cells were lysed and L1 proteins were precipitated with anti-FLAG antibody coupled to magnetic beads. A representative quantification of the precipitated SAMHD1 T592A signal is shown. HRP signals were quantified with AIDA Image analyzer software and normalized on bait signal and WCL input signal. c 293T cells were transfected with a L1 expression plasmid together with empty vector (pcDNA), SAMHD1 T592A, or MOV10. Cells were lysed 2 days posttransfection. Lysates were treated with either <t>RNaseOUT</t> or 15 μg/ml RNaseA prior to ORF1p-T7 precipitation using anti-T7 antibody coupled to magnetic beads. d 293T cells were transfected with empty vector (pcDNA), L1 expression vector and expression plasmids for SAMHD1 wt, T592A, or T592D. Cells were lysed 2 days posttransfection and SAMHD1 was precipitated with an anti-myc antibody coupled to magnetic beads. Subsequently, MLV-RT and LEAP-RT reactions were performed and amplification products were visualized on a 2% agarose gel. Input protein content was controlled by immunoblot. One out of three independent experiments is shown. WCL: Whole cell lysate, IP: immunoprecipitation
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    SAMHD1 interacts with LINE-1. a 293T cells were transfected with the L1 expression vector pAD2TE1 together with plasmids encoding wt SAMHD1, SAMHD1 T592A, or SAMHD1 T592D. Two days posttransfection, cells were lysed and ORF1p-T7 was precipitated using anti-T7 antibody coupled to magnetic beads. The precipitates were analyzed by immunoblot. SAMHD1-myc, T592A-myc, and T592D-myc signals were quantified using AIDA Image Analyzer software and normalized on the T7 signal in IP (bait) and the myc signal in WCL (input). b 293T cells were transfected with expression plasmids for L1 ORF1p-FLAG or ORF2p-3xFLAG together with empty vector (pcDNA) or SAMHD1 T592A. Two days posttransfection, cells were lysed and L1 proteins were precipitated with anti-FLAG antibody coupled to magnetic beads. A representative quantification of the precipitated SAMHD1 T592A signal is shown. HRP signals were quantified with AIDA Image analyzer software and normalized on bait signal and WCL input signal. c 293T cells were transfected with a L1 expression plasmid together with empty vector (pcDNA), SAMHD1 T592A, or MOV10. Cells were lysed 2 days posttransfection. Lysates were treated with either <t>RNaseOUT</t> or 15 μg/ml RNaseA prior to ORF1p-T7 precipitation using anti-T7 antibody coupled to magnetic beads. d 293T cells were transfected with empty vector (pcDNA), L1 expression vector and expression plasmids for SAMHD1 wt, T592A, or T592D. Cells were lysed 2 days posttransfection and SAMHD1 was precipitated with an anti-myc antibody coupled to magnetic beads. Subsequently, MLV-RT and LEAP-RT reactions were performed and amplification products were visualized on a 2% agarose gel. Input protein content was controlled by immunoblot. One out of three independent experiments is shown. WCL: Whole cell lysate, IP: immunoprecipitation
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    SAMHD1 interacts with LINE-1. a 293T cells were transfected with the L1 expression vector pAD2TE1 together with plasmids encoding wt SAMHD1, SAMHD1 T592A, or SAMHD1 T592D. Two days posttransfection, cells were lysed and ORF1p-T7 was precipitated using anti-T7 antibody coupled to magnetic beads. The precipitates were analyzed by immunoblot. SAMHD1-myc, T592A-myc, and T592D-myc signals were quantified using AIDA Image Analyzer software and normalized on the T7 signal in IP (bait) and the myc signal in WCL (input). b 293T cells were transfected with expression plasmids for L1 ORF1p-FLAG or ORF2p-3xFLAG together with empty vector (pcDNA) or SAMHD1 T592A. Two days posttransfection, cells were lysed and L1 proteins were precipitated with anti-FLAG antibody coupled to magnetic beads. A representative quantification of the precipitated SAMHD1 T592A signal is shown. HRP signals were quantified with AIDA Image analyzer software and normalized on bait signal and WCL input signal. c 293T cells were transfected with a L1 expression plasmid together with empty vector (pcDNA), SAMHD1 T592A, or MOV10. Cells were lysed 2 days posttransfection. Lysates were treated with either <t>RNaseOUT</t> or 15 μg/ml RNaseA prior to ORF1p-T7 precipitation using anti-T7 antibody coupled to magnetic beads. d 293T cells were transfected with empty vector (pcDNA), L1 expression vector and expression plasmids for SAMHD1 wt, T592A, or T592D. Cells were lysed 2 days posttransfection and SAMHD1 was precipitated with an anti-myc antibody coupled to magnetic beads. Subsequently, MLV-RT and LEAP-RT reactions were performed and amplification products were visualized on a 2% agarose gel. Input protein content was controlled by immunoblot. One out of three independent experiments is shown. WCL: Whole cell lysate, IP: immunoprecipitation
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    SAMHD1 interacts with LINE-1. a 293T cells were transfected with the L1 expression vector pAD2TE1 together with plasmids encoding wt SAMHD1, SAMHD1 T592A, or SAMHD1 T592D. Two days posttransfection, cells were lysed and ORF1p-T7 was precipitated using anti-T7 antibody coupled to magnetic beads. The precipitates were analyzed by immunoblot. SAMHD1-myc, T592A-myc, and T592D-myc signals were quantified using AIDA Image Analyzer software and normalized on the T7 signal in IP (bait) and the myc signal in WCL (input). b 293T cells were transfected with expression plasmids for L1 ORF1p-FLAG or ORF2p-3xFLAG together with empty vector (pcDNA) or SAMHD1 T592A. Two days posttransfection, cells were lysed and L1 proteins were precipitated with anti-FLAG antibody coupled to magnetic beads. A representative quantification of the precipitated SAMHD1 T592A signal is shown. HRP signals were quantified with AIDA Image analyzer software and normalized on bait signal and WCL input signal. c 293T cells were transfected with a L1 expression plasmid together with empty vector (pcDNA), SAMHD1 T592A, or MOV10. Cells were lysed 2 days posttransfection. Lysates were treated with either <t>RNaseOUT</t> or 15 μg/ml RNaseA prior to ORF1p-T7 precipitation using anti-T7 antibody coupled to magnetic beads. d 293T cells were transfected with empty vector (pcDNA), L1 expression vector and expression plasmids for SAMHD1 wt, T592A, or T592D. Cells were lysed 2 days posttransfection and SAMHD1 was precipitated with an anti-myc antibody coupled to magnetic beads. Subsequently, MLV-RT and LEAP-RT reactions were performed and amplification products were visualized on a 2% agarose gel. Input protein content was controlled by immunoblot. One out of three independent experiments is shown. WCL: Whole cell lysate, IP: immunoprecipitation
    Superscript Iii Rnaseout First Strand Synthesis System Supermix Kit, 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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    SAMHD1 interacts with LINE-1. a 293T cells were transfected with the L1 expression vector pAD2TE1 together with plasmids encoding wt SAMHD1, SAMHD1 T592A, or SAMHD1 T592D. Two days posttransfection, cells were lysed and ORF1p-T7 was precipitated using anti-T7 antibody coupled to magnetic beads. The precipitates were analyzed by immunoblot. SAMHD1-myc, T592A-myc, and T592D-myc signals were quantified using AIDA Image Analyzer software and normalized on the T7 signal in IP (bait) and the myc signal in WCL (input). b 293T cells were transfected with expression plasmids for L1 ORF1p-FLAG or ORF2p-3xFLAG together with empty vector (pcDNA) or SAMHD1 T592A. Two days posttransfection, cells were lysed and L1 proteins were precipitated with anti-FLAG antibody coupled to magnetic beads. A representative quantification of the precipitated SAMHD1 T592A signal is shown. HRP signals were quantified with AIDA Image analyzer software and normalized on bait signal and WCL input signal. c 293T cells were transfected with a L1 expression plasmid together with empty vector (pcDNA), SAMHD1 T592A, or MOV10. Cells were lysed 2 days posttransfection. Lysates were treated with either <t>RNaseOUT</t> or 15 μg/ml RNaseA prior to ORF1p-T7 precipitation using anti-T7 antibody coupled to magnetic beads. d 293T cells were transfected with empty vector (pcDNA), L1 expression vector and expression plasmids for SAMHD1 wt, T592A, or T592D. Cells were lysed 2 days posttransfection and SAMHD1 was precipitated with an anti-myc antibody coupled to magnetic beads. Subsequently, MLV-RT and LEAP-RT reactions were performed and amplification products were visualized on a 2% agarose gel. Input protein content was controlled by immunoblot. One out of three independent experiments is shown. WCL: Whole cell lysate, IP: immunoprecipitation
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    SAMHD1 interacts with LINE-1. a 293T cells were transfected with the L1 expression vector pAD2TE1 together with plasmids encoding wt SAMHD1, SAMHD1 T592A, or SAMHD1 T592D. Two days posttransfection, cells were lysed and ORF1p-T7 was precipitated using anti-T7 antibody coupled to magnetic beads. The precipitates were analyzed by immunoblot. SAMHD1-myc, T592A-myc, and T592D-myc signals were quantified using AIDA Image Analyzer software and normalized on the T7 signal in IP (bait) and the myc signal in WCL (input). b 293T cells were transfected with expression plasmids for L1 ORF1p-FLAG or ORF2p-3xFLAG together with empty vector (pcDNA) or SAMHD1 T592A. Two days posttransfection, cells were lysed and L1 proteins were precipitated with anti-FLAG antibody coupled to magnetic beads. A representative quantification of the precipitated SAMHD1 T592A signal is shown. HRP signals were quantified with AIDA Image analyzer software and normalized on bait signal and WCL input signal. c 293T cells were transfected with a L1 expression plasmid together with empty vector (pcDNA), SAMHD1 T592A, or MOV10. Cells were lysed 2 days posttransfection. Lysates were treated with either <t>RNaseOUT</t> or 15 μg/ml RNaseA prior to ORF1p-T7 precipitation using anti-T7 antibody coupled to magnetic beads. d 293T cells were transfected with empty vector (pcDNA), L1 expression vector and expression plasmids for SAMHD1 wt, T592A, or T592D. Cells were lysed 2 days posttransfection and SAMHD1 was precipitated with an anti-myc antibody coupled to magnetic beads. Subsequently, MLV-RT and LEAP-RT reactions were performed and amplification products were visualized on a 2% agarose gel. Input protein content was controlled by immunoblot. One out of three independent experiments is shown. WCL: Whole cell lysate, IP: immunoprecipitation
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    SAMHD1 interacts with LINE-1. a 293T cells were transfected with the L1 expression vector pAD2TE1 together with plasmids encoding wt SAMHD1, SAMHD1 T592A, or SAMHD1 T592D. Two days posttransfection, cells were lysed and ORF1p-T7 was precipitated using anti-T7 antibody coupled to magnetic beads. The precipitates were analyzed by immunoblot. SAMHD1-myc, T592A-myc, and T592D-myc signals were quantified using AIDA Image Analyzer software and normalized on the T7 signal in IP (bait) and the myc signal in WCL (input). b 293T cells were transfected with expression plasmids for L1 ORF1p-FLAG or ORF2p-3xFLAG together with empty vector (pcDNA) or SAMHD1 T592A. Two days posttransfection, cells were lysed and L1 proteins were precipitated with anti-FLAG antibody coupled to magnetic beads. A representative quantification of the precipitated SAMHD1 T592A signal is shown. HRP signals were quantified with AIDA Image analyzer software and normalized on bait signal and WCL input signal. c 293T cells were transfected with a L1 expression plasmid together with empty vector (pcDNA), SAMHD1 T592A, or MOV10. Cells were lysed 2 days posttransfection. Lysates were treated with either <t>RNaseOUT</t> or 15 μg/ml RNaseA prior to ORF1p-T7 precipitation using anti-T7 antibody coupled to magnetic beads. d 293T cells were transfected with empty vector (pcDNA), L1 expression vector and expression plasmids for SAMHD1 wt, T592A, or T592D. Cells were lysed 2 days posttransfection and SAMHD1 was precipitated with an anti-myc antibody coupled to magnetic beads. Subsequently, MLV-RT and LEAP-RT reactions were performed and amplification products were visualized on a 2% agarose gel. Input protein content was controlled by immunoblot. One out of three independent experiments is shown. WCL: Whole cell lysate, IP: immunoprecipitation
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    SAMHD1 interacts with LINE-1. a 293T cells were transfected with the L1 expression vector pAD2TE1 together with plasmids encoding wt SAMHD1, SAMHD1 T592A, or SAMHD1 T592D. Two days posttransfection, cells were lysed and ORF1p-T7 was precipitated using anti-T7 antibody coupled to magnetic beads. The precipitates were analyzed by immunoblot. SAMHD1-myc, T592A-myc, and T592D-myc signals were quantified using AIDA Image Analyzer software and normalized on the T7 signal in IP (bait) and the myc signal in WCL (input). b 293T cells were transfected with expression plasmids for L1 ORF1p-FLAG or ORF2p-3xFLAG together with empty vector (pcDNA) or SAMHD1 T592A. Two days posttransfection, cells were lysed and L1 proteins were precipitated with anti-FLAG antibody coupled to magnetic beads. A representative quantification of the precipitated SAMHD1 T592A signal is shown. HRP signals were quantified with AIDA Image analyzer software and normalized on bait signal and WCL input signal. c 293T cells were transfected with a L1 expression plasmid together with empty vector (pcDNA), SAMHD1 T592A, or MOV10. Cells were lysed 2 days posttransfection. Lysates were treated with either <t>RNaseOUT</t> or 15 μg/ml RNaseA prior to ORF1p-T7 precipitation using anti-T7 antibody coupled to magnetic beads. d 293T cells were transfected with empty vector (pcDNA), L1 expression vector and expression plasmids for SAMHD1 wt, T592A, or T592D. Cells were lysed 2 days posttransfection and SAMHD1 was precipitated with an anti-myc antibody coupled to magnetic beads. Subsequently, MLV-RT and LEAP-RT reactions were performed and amplification products were visualized on a 2% agarose gel. Input protein content was controlled by immunoblot. One out of three independent experiments is shown. WCL: Whole cell lysate, IP: immunoprecipitation
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    SAMHD1 interacts with LINE-1. a 293T cells were transfected with the L1 expression vector pAD2TE1 together with plasmids encoding wt SAMHD1, SAMHD1 T592A, or SAMHD1 T592D. Two days posttransfection, cells were lysed and ORF1p-T7 was precipitated using anti-T7 antibody coupled to magnetic beads. The precipitates were analyzed by immunoblot. SAMHD1-myc, T592A-myc, and T592D-myc signals were quantified using AIDA Image Analyzer software and normalized on the T7 signal in IP (bait) and the myc signal in WCL (input). b 293T cells were transfected with expression plasmids for L1 ORF1p-FLAG or ORF2p-3xFLAG together with empty vector (pcDNA) or SAMHD1 T592A. Two days posttransfection, cells were lysed and L1 proteins were precipitated with anti-FLAG antibody coupled to magnetic beads. A representative quantification of the precipitated SAMHD1 T592A signal is shown. HRP signals were quantified with AIDA Image analyzer software and normalized on bait signal and WCL input signal. c 293T cells were transfected with a L1 expression plasmid together with empty vector (pcDNA), SAMHD1 T592A, or MOV10. Cells were lysed 2 days posttransfection. Lysates were treated with either <t>RNaseOUT</t> or 15 μg/ml RNaseA prior to ORF1p-T7 precipitation using anti-T7 antibody coupled to magnetic beads. d 293T cells were transfected with empty vector (pcDNA), L1 expression vector and expression plasmids for SAMHD1 wt, T592A, or T592D. Cells were lysed 2 days posttransfection and SAMHD1 was precipitated with an anti-myc antibody coupled to magnetic beads. Subsequently, MLV-RT and LEAP-RT reactions were performed and amplification products were visualized on a 2% agarose gel. Input protein content was controlled by immunoblot. One out of three independent experiments is shown. WCL: Whole cell lysate, IP: immunoprecipitation
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    Thermo Fisher buffer b
    SAMHD1 interacts with LINE-1. a 293T cells were transfected with the L1 expression vector pAD2TE1 together with plasmids encoding wt SAMHD1, SAMHD1 T592A, or SAMHD1 T592D. Two days posttransfection, cells were lysed and ORF1p-T7 was precipitated using anti-T7 antibody coupled to magnetic beads. The precipitates were analyzed by immunoblot. SAMHD1-myc, T592A-myc, and T592D-myc signals were quantified using AIDA Image Analyzer software and normalized on the T7 signal in IP (bait) and the myc signal in WCL (input). b 293T cells were transfected with expression plasmids for L1 ORF1p-FLAG or ORF2p-3xFLAG together with empty vector (pcDNA) or SAMHD1 T592A. Two days posttransfection, cells were lysed and L1 proteins were precipitated with anti-FLAG antibody coupled to magnetic beads. A representative quantification of the precipitated SAMHD1 T592A signal is shown. HRP signals were quantified with AIDA Image analyzer software and normalized on bait signal and WCL input signal. c 293T cells were transfected with a L1 expression plasmid together with empty vector (pcDNA), SAMHD1 T592A, or MOV10. Cells were lysed 2 days posttransfection. Lysates were treated with either <t>RNaseOUT</t> or 15 μg/ml RNaseA prior to ORF1p-T7 precipitation using anti-T7 antibody coupled to magnetic beads. d 293T cells were transfected with empty vector (pcDNA), L1 expression vector and expression plasmids for SAMHD1 wt, T592A, or T592D. Cells were lysed 2 days posttransfection and SAMHD1 was precipitated with an anti-myc antibody coupled to magnetic beads. Subsequently, MLV-RT and LEAP-RT reactions were performed and amplification products were visualized on a 2% agarose gel. Input protein content was controlled by immunoblot. One out of three independent experiments is shown. WCL: Whole cell lysate, IP: immunoprecipitation
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    Thermo Fisher buffer c
    SAMHD1 interacts with LINE-1. a 293T cells were transfected with the L1 expression vector pAD2TE1 together with plasmids encoding wt SAMHD1, SAMHD1 T592A, or SAMHD1 T592D. Two days posttransfection, cells were lysed and ORF1p-T7 was precipitated using anti-T7 antibody coupled to magnetic beads. The precipitates were analyzed by immunoblot. SAMHD1-myc, T592A-myc, and T592D-myc signals were quantified using AIDA Image Analyzer software and normalized on the T7 signal in IP (bait) and the myc signal in WCL (input). b 293T cells were transfected with expression plasmids for L1 ORF1p-FLAG or ORF2p-3xFLAG together with empty vector (pcDNA) or SAMHD1 T592A. Two days posttransfection, cells were lysed and L1 proteins were precipitated with anti-FLAG antibody coupled to magnetic beads. A representative quantification of the precipitated SAMHD1 T592A signal is shown. HRP signals were quantified with AIDA Image analyzer software and normalized on bait signal and WCL input signal. c 293T cells were transfected with a L1 expression plasmid together with empty vector (pcDNA), SAMHD1 T592A, or MOV10. Cells were lysed 2 days posttransfection. Lysates were treated with either <t>RNaseOUT</t> or 15 μg/ml RNaseA prior to ORF1p-T7 precipitation using anti-T7 antibody coupled to magnetic beads. d 293T cells were transfected with empty vector (pcDNA), L1 expression vector and expression plasmids for SAMHD1 wt, T592A, or T592D. Cells were lysed 2 days posttransfection and SAMHD1 was precipitated with an anti-myc antibody coupled to magnetic beads. Subsequently, MLV-RT and LEAP-RT reactions were performed and amplification products were visualized on a 2% agarose gel. Input protein content was controlled by immunoblot. One out of three independent experiments is shown. WCL: Whole cell lysate, IP: immunoprecipitation
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    Thermo Fisher wash buffer
    SAMHD1 interacts with LINE-1. a 293T cells were transfected with the L1 expression vector pAD2TE1 together with plasmids encoding wt SAMHD1, SAMHD1 T592A, or SAMHD1 T592D. Two days posttransfection, cells were lysed and ORF1p-T7 was precipitated using anti-T7 antibody coupled to magnetic beads. The precipitates were analyzed by immunoblot. SAMHD1-myc, T592A-myc, and T592D-myc signals were quantified using AIDA Image Analyzer software and normalized on the T7 signal in IP (bait) and the myc signal in WCL (input). b 293T cells were transfected with expression plasmids for L1 ORF1p-FLAG or ORF2p-3xFLAG together with empty vector (pcDNA) or SAMHD1 T592A. Two days posttransfection, cells were lysed and L1 proteins were precipitated with anti-FLAG antibody coupled to magnetic beads. A representative quantification of the precipitated SAMHD1 T592A signal is shown. HRP signals were quantified with AIDA Image analyzer software and normalized on bait signal and WCL input signal. c 293T cells were transfected with a L1 expression plasmid together with empty vector (pcDNA), SAMHD1 T592A, or MOV10. Cells were lysed 2 days posttransfection. Lysates were treated with either <t>RNaseOUT</t> or 15 μg/ml RNaseA prior to ORF1p-T7 precipitation using anti-T7 antibody coupled to magnetic beads. d 293T cells were transfected with empty vector (pcDNA), L1 expression vector and expression plasmids for SAMHD1 wt, T592A, or T592D. Cells were lysed 2 days posttransfection and SAMHD1 was precipitated with an anti-myc antibody coupled to magnetic beads. Subsequently, MLV-RT and LEAP-RT reactions were performed and amplification products were visualized on a 2% agarose gel. Input protein content was controlled by immunoblot. One out of three independent experiments is shown. WCL: Whole cell lysate, IP: immunoprecipitation
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    Thermo Fisher transcription rt pcr mastermix buffer
    SAMHD1 interacts with LINE-1. a 293T cells were transfected with the L1 expression vector pAD2TE1 together with plasmids encoding wt SAMHD1, SAMHD1 T592A, or SAMHD1 T592D. Two days posttransfection, cells were lysed and ORF1p-T7 was precipitated using anti-T7 antibody coupled to magnetic beads. The precipitates were analyzed by immunoblot. SAMHD1-myc, T592A-myc, and T592D-myc signals were quantified using AIDA Image Analyzer software and normalized on the T7 signal in IP (bait) and the myc signal in WCL (input). b 293T cells were transfected with expression plasmids for L1 ORF1p-FLAG or ORF2p-3xFLAG together with empty vector (pcDNA) or SAMHD1 T592A. Two days posttransfection, cells were lysed and L1 proteins were precipitated with anti-FLAG antibody coupled to magnetic beads. A representative quantification of the precipitated SAMHD1 T592A signal is shown. HRP signals were quantified with AIDA Image analyzer software and normalized on bait signal and WCL input signal. c 293T cells were transfected with a L1 expression plasmid together with empty vector (pcDNA), SAMHD1 T592A, or MOV10. Cells were lysed 2 days posttransfection. Lysates were treated with either <t>RNaseOUT</t> or 15 μg/ml RNaseA prior to ORF1p-T7 precipitation using anti-T7 antibody coupled to magnetic beads. d 293T cells were transfected with empty vector (pcDNA), L1 expression vector and expression plasmids for SAMHD1 wt, T592A, or T592D. Cells were lysed 2 days posttransfection and SAMHD1 was precipitated with an anti-myc antibody coupled to magnetic beads. Subsequently, MLV-RT and LEAP-RT reactions were performed and amplification products were visualized on a 2% agarose gel. Input protein content was controlled by immunoblot. One out of three independent experiments is shown. WCL: Whole cell lysate, IP: immunoprecipitation
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    Image Search Results


    SAMHD1 interacts with LINE-1. a 293T cells were transfected with the L1 expression vector pAD2TE1 together with plasmids encoding wt SAMHD1, SAMHD1 T592A, or SAMHD1 T592D. Two days posttransfection, cells were lysed and ORF1p-T7 was precipitated using anti-T7 antibody coupled to magnetic beads. The precipitates were analyzed by immunoblot. SAMHD1-myc, T592A-myc, and T592D-myc signals were quantified using AIDA Image Analyzer software and normalized on the T7 signal in IP (bait) and the myc signal in WCL (input). b 293T cells were transfected with expression plasmids for L1 ORF1p-FLAG or ORF2p-3xFLAG together with empty vector (pcDNA) or SAMHD1 T592A. Two days posttransfection, cells were lysed and L1 proteins were precipitated with anti-FLAG antibody coupled to magnetic beads. A representative quantification of the precipitated SAMHD1 T592A signal is shown. HRP signals were quantified with AIDA Image analyzer software and normalized on bait signal and WCL input signal. c 293T cells were transfected with a L1 expression plasmid together with empty vector (pcDNA), SAMHD1 T592A, or MOV10. Cells were lysed 2 days posttransfection. Lysates were treated with either RNaseOUT or 15 μg/ml RNaseA prior to ORF1p-T7 precipitation using anti-T7 antibody coupled to magnetic beads. d 293T cells were transfected with empty vector (pcDNA), L1 expression vector and expression plasmids for SAMHD1 wt, T592A, or T592D. Cells were lysed 2 days posttransfection and SAMHD1 was precipitated with an anti-myc antibody coupled to magnetic beads. Subsequently, MLV-RT and LEAP-RT reactions were performed and amplification products were visualized on a 2% agarose gel. Input protein content was controlled by immunoblot. One out of three independent experiments is shown. WCL: Whole cell lysate, IP: immunoprecipitation

    Journal: Mobile DNA

    Article Title: The SAMHD1-mediated block of LINE-1 retroelements is regulated by phosphorylation

    doi: 10.1186/s13100-018-0116-5

    Figure Lengend Snippet: SAMHD1 interacts with LINE-1. a 293T cells were transfected with the L1 expression vector pAD2TE1 together with plasmids encoding wt SAMHD1, SAMHD1 T592A, or SAMHD1 T592D. Two days posttransfection, cells were lysed and ORF1p-T7 was precipitated using anti-T7 antibody coupled to magnetic beads. The precipitates were analyzed by immunoblot. SAMHD1-myc, T592A-myc, and T592D-myc signals were quantified using AIDA Image Analyzer software and normalized on the T7 signal in IP (bait) and the myc signal in WCL (input). b 293T cells were transfected with expression plasmids for L1 ORF1p-FLAG or ORF2p-3xFLAG together with empty vector (pcDNA) or SAMHD1 T592A. Two days posttransfection, cells were lysed and L1 proteins were precipitated with anti-FLAG antibody coupled to magnetic beads. A representative quantification of the precipitated SAMHD1 T592A signal is shown. HRP signals were quantified with AIDA Image analyzer software and normalized on bait signal and WCL input signal. c 293T cells were transfected with a L1 expression plasmid together with empty vector (pcDNA), SAMHD1 T592A, or MOV10. Cells were lysed 2 days posttransfection. Lysates were treated with either RNaseOUT or 15 μg/ml RNaseA prior to ORF1p-T7 precipitation using anti-T7 antibody coupled to magnetic beads. d 293T cells were transfected with empty vector (pcDNA), L1 expression vector and expression plasmids for SAMHD1 wt, T592A, or T592D. Cells were lysed 2 days posttransfection and SAMHD1 was precipitated with an anti-myc antibody coupled to magnetic beads. Subsequently, MLV-RT and LEAP-RT reactions were performed and amplification products were visualized on a 2% agarose gel. Input protein content was controlled by immunoblot. One out of three independent experiments is shown. WCL: Whole cell lysate, IP: immunoprecipitation

    Article Snippet: Immunoprecipitation assays 293T cells were lysed 48 h posttransfection in 160 mM NaCl, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 0.25% NP-40, supplemented with Halt Protease Inhibitor, 1 mM PMSF (Sigma), and RNaseOUT (Life Technologies).

    Techniques: Transfection, Expressing, Plasmid Preparation, Magnetic Beads, Software, Amplification, Agarose Gel Electrophoresis, Immunoprecipitation

    Eudicot S-RNase protein conserved domain structure. The conserved domain structures of S-RNases employed in GSI Eudicot lineages and the conserved domains revealed in this study of Coffea are labeled accordingly. Canonical S-RNase conserved regions are labeled “c1”–“c5”, and hyper-variable regions are labeled “HVa” and “HVb”. The red arrowheads below the protein constructs indicate intron positions. Intron position data and presence of conserved region c1 was validated in Coffea based on genomic sequence and 5′RACE data from C. arabica and C. canephora alone (data not shown). Results relating to RNase polymorphism reported in this study are based on 3′RACE data from the hyper-variable regions (i.e. HVa) to the end of transcripts (i.e. through c5). This figure was adapted from Vieira and Charlesworth [91] .

    Journal: PLoS ONE

    Article Title: Expression and Trans-Specific Polymorphism of Self-Incompatibility RNases in Coffea (Rubiaceae)

    doi: 10.1371/journal.pone.0021019

    Figure Lengend Snippet: Eudicot S-RNase protein conserved domain structure. The conserved domain structures of S-RNases employed in GSI Eudicot lineages and the conserved domains revealed in this study of Coffea are labeled accordingly. Canonical S-RNase conserved regions are labeled “c1”–“c5”, and hyper-variable regions are labeled “HVa” and “HVb”. The red arrowheads below the protein constructs indicate intron positions. Intron position data and presence of conserved region c1 was validated in Coffea based on genomic sequence and 5′RACE data from C. arabica and C. canephora alone (data not shown). Results relating to RNase polymorphism reported in this study are based on 3′RACE data from the hyper-variable regions (i.e. HVa) to the end of transcripts (i.e. through c5). This figure was adapted from Vieira and Charlesworth [91] .

    Article Snippet: Briefly, first strand cDNA was synthesized from heat-denatured (80°C for 5 mins) total RNA (150 ng – 1 µg) with MMLV reverse transcriptase (Promega), RNase inhibitor (RNaseOut, Invitrogen), and a 3′ RACE adapter primer at 37°C for one hour.

    Techniques: Labeling, Construct, Sequencing

    Expression of RNase T2 genes in C. arabica tissues. Agarose gel image showing RT-PCR results using gene-specific primers for Coffea RNase “A”, “C”, and S-RNase genes. cDNAs were generated from RNA extracts of various C. arabica tissues labeled as follows: P1 = pre-anthesis pistils dissected from flower buds; P2 = post-anthesis pistils; L1 = leaf at 1 week; L2 = leaf at > 2 weeks; R1 = fine root; R2 = primary root; F1 = fruit at approximately 1 week; F2 = fruit at > 3 weeks. RNase “A” and “C” genes are constitutively expressed in C. arabica tissues, but the S-RNase gene shows pistil-specific expression characteristic of genes involved in the S-RNase GSI system.

    Journal: PLoS ONE

    Article Title: Expression and Trans-Specific Polymorphism of Self-Incompatibility RNases in Coffea (Rubiaceae)

    doi: 10.1371/journal.pone.0021019

    Figure Lengend Snippet: Expression of RNase T2 genes in C. arabica tissues. Agarose gel image showing RT-PCR results using gene-specific primers for Coffea RNase “A”, “C”, and S-RNase genes. cDNAs were generated from RNA extracts of various C. arabica tissues labeled as follows: P1 = pre-anthesis pistils dissected from flower buds; P2 = post-anthesis pistils; L1 = leaf at 1 week; L2 = leaf at > 2 weeks; R1 = fine root; R2 = primary root; F1 = fruit at approximately 1 week; F2 = fruit at > 3 weeks. RNase “A” and “C” genes are constitutively expressed in C. arabica tissues, but the S-RNase gene shows pistil-specific expression characteristic of genes involved in the S-RNase GSI system.

    Article Snippet: Briefly, first strand cDNA was synthesized from heat-denatured (80°C for 5 mins) total RNA (150 ng – 1 µg) with MMLV reverse transcriptase (Promega), RNase inhibitor (RNaseOut, Invitrogen), and a 3′ RACE adapter primer at 37°C for one hour.

    Techniques: Expressing, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Generated, Labeling

    Kinetics and sensitivity of purified RNA CHA circuit. ( a ) Fold amplification and sensitivity of gel-purified RNA CHA circuit. The RNA CHA circuit can detect pure C1 to picomolar concentration with ∼87-fold amplification of 0.1 nM C1 within 315 min at 52°C. Circuit output measured as concentration of RepF released from RepF:RepQ duplex was extrapolated from a standard curve of free RepF. ( b ) Initial rate of C1-catalyzed H1:H2 hybridization was measured by incubating varying concentrations of gel-purified H1 and H2 with 2.5 nM pure C1 RNA diluted in 1 µM oligo dT 17 . Circuits were executed in 1× TNaK buffer containing 20 U of RNaseOUT, 0.5 µM ROX reference dye and 400 nM RepF annealed with 5× excess (2 µM) RepQ at 52°C for 315 min. Initial rates were calculated from circuit output measurements made during the initial 3–20 min of circuit operation. Average data from three separate experiments are represented. H1 concentration has a greater impact on the initial rate suggesting that the first step of the circuit (C1-triggered unfolding of H1) is a rate limiting step. ( c ) Effect of H1 and H2 concentrations on the kinetics of RNA CHA circuit. Average raw fluorescence data from triplicate experiments are plotted. Circuit output is maximal when operated with near equal concentrations of H1 and H2. Increasing H2 concentration above that of H1 generally decreased the initial reaction rate and resulted in reduced circuit output.

    Journal: Nucleic Acids Research

    Article Title: Design and application of cotranscriptional non-enzymatic RNA circuits and signal transducers

    doi: 10.1093/nar/gku074

    Figure Lengend Snippet: Kinetics and sensitivity of purified RNA CHA circuit. ( a ) Fold amplification and sensitivity of gel-purified RNA CHA circuit. The RNA CHA circuit can detect pure C1 to picomolar concentration with ∼87-fold amplification of 0.1 nM C1 within 315 min at 52°C. Circuit output measured as concentration of RepF released from RepF:RepQ duplex was extrapolated from a standard curve of free RepF. ( b ) Initial rate of C1-catalyzed H1:H2 hybridization was measured by incubating varying concentrations of gel-purified H1 and H2 with 2.5 nM pure C1 RNA diluted in 1 µM oligo dT 17 . Circuits were executed in 1× TNaK buffer containing 20 U of RNaseOUT, 0.5 µM ROX reference dye and 400 nM RepF annealed with 5× excess (2 µM) RepQ at 52°C for 315 min. Initial rates were calculated from circuit output measurements made during the initial 3–20 min of circuit operation. Average data from three separate experiments are represented. H1 concentration has a greater impact on the initial rate suggesting that the first step of the circuit (C1-triggered unfolding of H1) is a rate limiting step. ( c ) Effect of H1 and H2 concentrations on the kinetics of RNA CHA circuit. Average raw fluorescence data from triplicate experiments are plotted. Circuit output is maximal when operated with near equal concentrations of H1 and H2. Increasing H2 concentration above that of H1 generally decreased the initial reaction rate and resulted in reduced circuit output.

    Article Snippet: In vitro transcription In all, 100 pg to 1000 ng of double-stranded DNA transcription templates was transcribed using 100 U of T7 RNA polymerase (NEB) in 50 µl reactions containing 40 mM Tris–HCl, pH 7.9, 30 mM MgCl2 , 10 mM DTT, 2 mM spermidine, 4 mM ribonucleotide (rNTP) mix and 20 U of the recombinant ribonuclease inhibitor RNaseOUT (Life Technologies).

    Techniques: Purification, Amplification, Concentration Assay, Hybridization, Fluorescence

    Operation of cotranscriptionally generated RNA CHA circuits without any downstream purification and design optimization for detection of DNA target. ( a ) Fifty nanograms each of the indicated pairs of hairpin 1 and 2 transcription templates was cotranscribed with or without 10 ng of C1 transcription template for 1 h at 42°C using T7 RNA polymerase. Following transcription, 2 µl of the reaction mix was directly incubated in 1× TNaK buffer containing 20 U of RNaseOUT and 0.5 µM ROX reference dye along with 400 nM RepF (annealed with 5× excess RepQ) fluorescent DNA reporter duplex for quantitating RNA CHA in real-time at 52°C. ( b ) Schematic depicting SDA of DNA. The single-stranded template DNA (black arrow) consists of a sequence (C*) complementary to the RNA CHA catalyst followed by the nicking enzyme recognition sequence (NE) that is present on the non-cleaved DNA strand and a primer binding site. Following primer binding (step 1), the DNA polymerase synthesizes the complementary strand that now completes the duplex NE site and contains the RNA CHA catalyst sequence (C). Nicking enzyme then binds the duplex NE site (step 2) and cleaves the newly synthesized strand at the NE site. The new 3′-OH group generated at the nick site is then extended by the DNA polymerase (step 3) while displacing the previously synthesized strand. The displaced ssDNA amplicon can then catalyze RNA CHA. ( c ) Schematic of DNA target sequence design for catalysis of RNA CHA. Single toehold (domain 1*) DNA target C1 (generated by SDA from the template TLTRSDA) with the same domain architecture as the RNA C1 is an inefficient catalyst of RNA CHA. Extended DNA target C1234 (generated by SDA from the template 1234LTRSDA) presenting two toeholds for RNA H1 successfully catalyzes RNA CHA.

    Journal: Nucleic Acids Research

    Article Title: Design and application of cotranscriptional non-enzymatic RNA circuits and signal transducers

    doi: 10.1093/nar/gku074

    Figure Lengend Snippet: Operation of cotranscriptionally generated RNA CHA circuits without any downstream purification and design optimization for detection of DNA target. ( a ) Fifty nanograms each of the indicated pairs of hairpin 1 and 2 transcription templates was cotranscribed with or without 10 ng of C1 transcription template for 1 h at 42°C using T7 RNA polymerase. Following transcription, 2 µl of the reaction mix was directly incubated in 1× TNaK buffer containing 20 U of RNaseOUT and 0.5 µM ROX reference dye along with 400 nM RepF (annealed with 5× excess RepQ) fluorescent DNA reporter duplex for quantitating RNA CHA in real-time at 52°C. ( b ) Schematic depicting SDA of DNA. The single-stranded template DNA (black arrow) consists of a sequence (C*) complementary to the RNA CHA catalyst followed by the nicking enzyme recognition sequence (NE) that is present on the non-cleaved DNA strand and a primer binding site. Following primer binding (step 1), the DNA polymerase synthesizes the complementary strand that now completes the duplex NE site and contains the RNA CHA catalyst sequence (C). Nicking enzyme then binds the duplex NE site (step 2) and cleaves the newly synthesized strand at the NE site. The new 3′-OH group generated at the nick site is then extended by the DNA polymerase (step 3) while displacing the previously synthesized strand. The displaced ssDNA amplicon can then catalyze RNA CHA. ( c ) Schematic of DNA target sequence design for catalysis of RNA CHA. Single toehold (domain 1*) DNA target C1 (generated by SDA from the template TLTRSDA) with the same domain architecture as the RNA C1 is an inefficient catalyst of RNA CHA. Extended DNA target C1234 (generated by SDA from the template 1234LTRSDA) presenting two toeholds for RNA H1 successfully catalyzes RNA CHA.

    Article Snippet: In vitro transcription In all, 100 pg to 1000 ng of double-stranded DNA transcription templates was transcribed using 100 U of T7 RNA polymerase (NEB) in 50 µl reactions containing 40 mM Tris–HCl, pH 7.9, 30 mM MgCl2 , 10 mM DTT, 2 mM spermidine, 4 mM ribonucleotide (rNTP) mix and 20 U of the recombinant ribonuclease inhibitor RNaseOUT (Life Technologies).

    Techniques: Generated, Purification, Incubation, Sequencing, Binding Assay, Synthesized, Amplification

    Synthesis and execution of RNA CHA circuit. ( a ) LHRz and RHRz-mediated cotranscriptional RNA cleavage releases the internal circuit components H1, H2 and C1. Fifity nanograms of PCR-generated transcription templates for H1, H2 and C1 was transcribed in 50 µl of reactions by T7 RNA polymerase for 2 h at 42°C. Two microliters of the resulting transcripts was analyzed by electrophoresis on a 10% denaturing polyacrylamide gel. Single-stranded DNA oligonucleotides were used as size markers. ( b ) RNA hairpins undergo catalyzed assembly into RNA duplexes. Gel-purified RNA catalyst C1 and the hairpins H1 and H2 were combined as indicated and incubated in 1× TNaK buffer containing 20 U of RNaseOUT for 150 min at 42°C (lanes 1–4), 52°C (lanes 5–8) or 62°C (lanes 9–12). The reactions were then analyzed on a 10% native polyacrylamide gel. Fifteen nanograms of C1 RNA was included in lane 13 as a control. Single-stranded DNA oligonucleotides were used as size markers.

    Journal: Nucleic Acids Research

    Article Title: Design and application of cotranscriptional non-enzymatic RNA circuits and signal transducers

    doi: 10.1093/nar/gku074

    Figure Lengend Snippet: Synthesis and execution of RNA CHA circuit. ( a ) LHRz and RHRz-mediated cotranscriptional RNA cleavage releases the internal circuit components H1, H2 and C1. Fifity nanograms of PCR-generated transcription templates for H1, H2 and C1 was transcribed in 50 µl of reactions by T7 RNA polymerase for 2 h at 42°C. Two microliters of the resulting transcripts was analyzed by electrophoresis on a 10% denaturing polyacrylamide gel. Single-stranded DNA oligonucleotides were used as size markers. ( b ) RNA hairpins undergo catalyzed assembly into RNA duplexes. Gel-purified RNA catalyst C1 and the hairpins H1 and H2 were combined as indicated and incubated in 1× TNaK buffer containing 20 U of RNaseOUT for 150 min at 42°C (lanes 1–4), 52°C (lanes 5–8) or 62°C (lanes 9–12). The reactions were then analyzed on a 10% native polyacrylamide gel. Fifteen nanograms of C1 RNA was included in lane 13 as a control. Single-stranded DNA oligonucleotides were used as size markers.

    Article Snippet: In vitro transcription In all, 100 pg to 1000 ng of double-stranded DNA transcription templates was transcribed using 100 U of T7 RNA polymerase (NEB) in 50 µl reactions containing 40 mM Tris–HCl, pH 7.9, 30 mM MgCl2 , 10 mM DTT, 2 mM spermidine, 4 mM ribonucleotide (rNTP) mix and 20 U of the recombinant ribonuclease inhibitor RNaseOUT (Life Technologies).

    Techniques: Polymerase Chain Reaction, Generated, Electrophoresis, Purification, Incubation

    Cotranscriptionally generated RNA CHA as signal transducer for nucleic acid diagnostics. ( a ) End-point sequence-specific detection of SDA-generated ssDNA targets by RNA CHA. Samples with or without 10 nM template 1234LTRSDA were amplified by SDA for 90 min at 37°C in 25 µl of reaction volumes. Reactions were then incubated at 95°C for 5 min and stored at room temperature before assay by RNA CHA. Five microliters of these SDA products was then probed with 2 µl of Sephadex G25 column-purified cotranscribed mH1:H2 RNA CHA circuit. RNA CHA cotranscriptions were performed with T7 RNA polymerase using 50 ng each of the mH1 and H2 transcription templates for 1 h at 42°C. End-point RNA CHA detection reactions were assembled in 1× TNaK buffer containing 20 U of RNaseOUT, 0.5 µM ROX reference dye and 100 nM RepF (annealed with 5× excess RepQ) fluorescent DNA reporter duplex for quantitating RNA CHA in real-time at 52°C. Negative control reactions lacking RNA CHA components or containing 2 µl of either only mH1 or H2 were also tested. ( b ) Real-time signal transduction of ssDNA-generating SDA by cotranscribed mH1:H2 RNA CHA. High temperature (55°C) SDA reactions were set up with or without 10 nM 1234HTRSDA template in 20 µl of volume containing 0.5 µM ROX reference dye and 75 nM RepF (annealed with 5× excess RepQ) fluorescent DNA reporter duplex for quantitating RNA CHA in real-time. Real-time sequence-specific signal transduction was achieved by adding 2 µl of unpurified mH1:H2 RNA CHA circuits cotranscribed from 50 ng of each transcription template to the SDA reactions. Control SDA reactions containing no RNA CHA components or 2 µl of either only mH1 or H2 were also tested.

    Journal: Nucleic Acids Research

    Article Title: Design and application of cotranscriptional non-enzymatic RNA circuits and signal transducers

    doi: 10.1093/nar/gku074

    Figure Lengend Snippet: Cotranscriptionally generated RNA CHA as signal transducer for nucleic acid diagnostics. ( a ) End-point sequence-specific detection of SDA-generated ssDNA targets by RNA CHA. Samples with or without 10 nM template 1234LTRSDA were amplified by SDA for 90 min at 37°C in 25 µl of reaction volumes. Reactions were then incubated at 95°C for 5 min and stored at room temperature before assay by RNA CHA. Five microliters of these SDA products was then probed with 2 µl of Sephadex G25 column-purified cotranscribed mH1:H2 RNA CHA circuit. RNA CHA cotranscriptions were performed with T7 RNA polymerase using 50 ng each of the mH1 and H2 transcription templates for 1 h at 42°C. End-point RNA CHA detection reactions were assembled in 1× TNaK buffer containing 20 U of RNaseOUT, 0.5 µM ROX reference dye and 100 nM RepF (annealed with 5× excess RepQ) fluorescent DNA reporter duplex for quantitating RNA CHA in real-time at 52°C. Negative control reactions lacking RNA CHA components or containing 2 µl of either only mH1 or H2 were also tested. ( b ) Real-time signal transduction of ssDNA-generating SDA by cotranscribed mH1:H2 RNA CHA. High temperature (55°C) SDA reactions were set up with or without 10 nM 1234HTRSDA template in 20 µl of volume containing 0.5 µM ROX reference dye and 75 nM RepF (annealed with 5× excess RepQ) fluorescent DNA reporter duplex for quantitating RNA CHA in real-time. Real-time sequence-specific signal transduction was achieved by adding 2 µl of unpurified mH1:H2 RNA CHA circuits cotranscribed from 50 ng of each transcription template to the SDA reactions. Control SDA reactions containing no RNA CHA components or 2 µl of either only mH1 or H2 were also tested.

    Article Snippet: In vitro transcription In all, 100 pg to 1000 ng of double-stranded DNA transcription templates was transcribed using 100 U of T7 RNA polymerase (NEB) in 50 µl reactions containing 40 mM Tris–HCl, pH 7.9, 30 mM MgCl2 , 10 mM DTT, 2 mM spermidine, 4 mM ribonucleotide (rNTP) mix and 20 U of the recombinant ribonuclease inhibitor RNaseOUT (Life Technologies).

    Techniques: Generated, Sequencing, Amplification, Incubation, Purification, Negative Control, Transduction

    Application of RNA CHA circuit as an OR logic processor. ( a ) Schematic of RNA CHA circuit operation in response to either catalyst C1 OR C2. The RNA hairpin H1B serves as the OR gate, and circuit output is measured fluorimetrically using Spinach.ST1 RNA aptamer beacon. ( b ) Circuit components (H1B and H2 RNA hairpins), reporter RNA (Spinach.ST1) and the inputs C1 and C2 were transcribed from 500 ng of duplex DNA transcription templates using T7 RNA polymerase. Transcription templates were prepared using the same procedure as Figure 8 . Following filtration through Sephadex G25, 3 µl/transcript (or 1.5 µl each of C1 and C2 when added together in a reaction) was mixed in the indicated combinations in 1× TNaK buffer containing 70 µM DFHBI and 20 U of RNaseOUT. Circuits were operated at 37°C, and outputs were measured fluorimetrically.

    Journal: Nucleic Acids Research

    Article Title: Design and application of cotranscriptional non-enzymatic RNA circuits and signal transducers

    doi: 10.1093/nar/gku074

    Figure Lengend Snippet: Application of RNA CHA circuit as an OR logic processor. ( a ) Schematic of RNA CHA circuit operation in response to either catalyst C1 OR C2. The RNA hairpin H1B serves as the OR gate, and circuit output is measured fluorimetrically using Spinach.ST1 RNA aptamer beacon. ( b ) Circuit components (H1B and H2 RNA hairpins), reporter RNA (Spinach.ST1) and the inputs C1 and C2 were transcribed from 500 ng of duplex DNA transcription templates using T7 RNA polymerase. Transcription templates were prepared using the same procedure as Figure 8 . Following filtration through Sephadex G25, 3 µl/transcript (or 1.5 µl each of C1 and C2 when added together in a reaction) was mixed in the indicated combinations in 1× TNaK buffer containing 70 µM DFHBI and 20 U of RNaseOUT. Circuits were operated at 37°C, and outputs were measured fluorimetrically.

    Article Snippet: In vitro transcription In all, 100 pg to 1000 ng of double-stranded DNA transcription templates was transcribed using 100 U of T7 RNA polymerase (NEB) in 50 µl reactions containing 40 mM Tris–HCl, pH 7.9, 30 mM MgCl2 , 10 mM DTT, 2 mM spermidine, 4 mM ribonucleotide (rNTP) mix and 20 U of the recombinant ribonuclease inhibitor RNaseOUT (Life Technologies).

    Techniques: Filtration

    Cotranscriptional RNA CHA and circuit design optimization for cotranscription. ( a ) Cotranscribed RNA circuit components undergo catalyzed hairpin assembly without requiring gel purification of individual reactants. Fifty nanograms each of H1 and H2 transcription templates, along with titrating amounts of C1 transcription template, was cotranscribed for 1 h at 42°C using T7 RNA polymerase followed by passage through Illustra MicroSpin Sephadex G25 columns. Transcription templates were amplified from cloned inserts using primers pCR2.1.F and pCR2.1.R specific to plasmid sequences flanking the inserts. Two microliter aliquots of the cotranscribed RNA mixtures were then incubated in 15 µl of volume with 400 nM RepF annealed with 5× excess (2 µM) RepQ fluorescent DNA reporter duplex in 1× TNaK buffer containing 20 U of RNaseOUT and 0.5 µM ROX reference dye to quantitate formation of H1:H2 RNA duplexes at 52°C. Average data from triplicate experiments are represented. ( b and c ) Schematic depicting sequences of RNA hairpins H1 and H2 with one- or two-base engineered mismatches. Mismatched H1 (mH1) presents a two-base mismatch between its domain 4* and domain 4 of H2. The hairpins mAH1 and mGH1 each contain a single mismatched base between their domain 4* and the domain 4 of H2. The mutated H2 hairpin m2H2 presents two mismatched bases between its domain 2* and the H1 domain 2.

    Journal: Nucleic Acids Research

    Article Title: Design and application of cotranscriptional non-enzymatic RNA circuits and signal transducers

    doi: 10.1093/nar/gku074

    Figure Lengend Snippet: Cotranscriptional RNA CHA and circuit design optimization for cotranscription. ( a ) Cotranscribed RNA circuit components undergo catalyzed hairpin assembly without requiring gel purification of individual reactants. Fifty nanograms each of H1 and H2 transcription templates, along with titrating amounts of C1 transcription template, was cotranscribed for 1 h at 42°C using T7 RNA polymerase followed by passage through Illustra MicroSpin Sephadex G25 columns. Transcription templates were amplified from cloned inserts using primers pCR2.1.F and pCR2.1.R specific to plasmid sequences flanking the inserts. Two microliter aliquots of the cotranscribed RNA mixtures were then incubated in 15 µl of volume with 400 nM RepF annealed with 5× excess (2 µM) RepQ fluorescent DNA reporter duplex in 1× TNaK buffer containing 20 U of RNaseOUT and 0.5 µM ROX reference dye to quantitate formation of H1:H2 RNA duplexes at 52°C. Average data from triplicate experiments are represented. ( b and c ) Schematic depicting sequences of RNA hairpins H1 and H2 with one- or two-base engineered mismatches. Mismatched H1 (mH1) presents a two-base mismatch between its domain 4* and domain 4 of H2. The hairpins mAH1 and mGH1 each contain a single mismatched base between their domain 4* and the domain 4 of H2. The mutated H2 hairpin m2H2 presents two mismatched bases between its domain 2* and the H1 domain 2.

    Article Snippet: In vitro transcription In all, 100 pg to 1000 ng of double-stranded DNA transcription templates was transcribed using 100 U of T7 RNA polymerase (NEB) in 50 µl reactions containing 40 mM Tris–HCl, pH 7.9, 30 mM MgCl2 , 10 mM DTT, 2 mM spermidine, 4 mM ribonucleotide (rNTP) mix and 20 U of the recombinant ribonuclease inhibitor RNaseOUT (Life Technologies).

    Techniques: Gel Purification, Amplification, Clone Assay, Plasmid Preparation, Incubation

    An entirely RNA-based CHA circuit operation and fluorimetric detection. ( a ) CHA circuit components (hairpins H1B and H2 and catalyst C1) and the RNA reporter Spinach.ST1 were separately transcribed by T7 RNA polymerase from 500 ng of PCR-generated duplex DNA transcription templates. H1B, H2 and C1 transcription templates were amplified using primers complementary to the exact ends of the cloned inserts (H1B.amp.F:H1B.amp.R, H2.amp.F:H2.amp.R and C1.amp.F:C1.amp.R, respectively) rather than the flanking plasmid. Spinach.ST transcription templates were amplified using primers specific to the flanking plasmid sequence at the 5′-end (pCR2.1.F) and the primer sphT.U.R specific to the 3′-end sequence of Spinach.ST. Transcription reactions were filtered through Sephadex G25 columns before circuit assembly. Three microliters of H1B, H2, C1 and Spinach.ST1 transcripts was mixed in indicated combinations and incubated in 1× TNaK buffer containing 70 µM DFHBI and 20 U of RNaseOUT. Circuit output was measured as increasing fluorescence intensity over time at 37°C. ( b–d ) Performance of DNA reporter duplex H1BF:H1BQ (b) versus Spinach.ST1 (c) in measuring RNA CHA circuit output. Indicated concentrations of gel-purified RNA hairpins H1B and H2 were incubated with equal concentration of H1BF:H1BQ or gel-purified Spinach.ST1 (+ 70 µM DFHBI) in the presence of titrating concentrations of pure C1 RNA. All circuits were operated in 1× TNaK buffer containing 20 U of RNaseOUT at 37°C, and average data from triplicate experiments are represented. Signal-to-noise ratio of H1BF:H1BQ versus Spinach.ST1 over the time course of RNA CHA detection is plotted in (d).

    Journal: Nucleic Acids Research

    Article Title: Design and application of cotranscriptional non-enzymatic RNA circuits and signal transducers

    doi: 10.1093/nar/gku074

    Figure Lengend Snippet: An entirely RNA-based CHA circuit operation and fluorimetric detection. ( a ) CHA circuit components (hairpins H1B and H2 and catalyst C1) and the RNA reporter Spinach.ST1 were separately transcribed by T7 RNA polymerase from 500 ng of PCR-generated duplex DNA transcription templates. H1B, H2 and C1 transcription templates were amplified using primers complementary to the exact ends of the cloned inserts (H1B.amp.F:H1B.amp.R, H2.amp.F:H2.amp.R and C1.amp.F:C1.amp.R, respectively) rather than the flanking plasmid. Spinach.ST transcription templates were amplified using primers specific to the flanking plasmid sequence at the 5′-end (pCR2.1.F) and the primer sphT.U.R specific to the 3′-end sequence of Spinach.ST. Transcription reactions were filtered through Sephadex G25 columns before circuit assembly. Three microliters of H1B, H2, C1 and Spinach.ST1 transcripts was mixed in indicated combinations and incubated in 1× TNaK buffer containing 70 µM DFHBI and 20 U of RNaseOUT. Circuit output was measured as increasing fluorescence intensity over time at 37°C. ( b–d ) Performance of DNA reporter duplex H1BF:H1BQ (b) versus Spinach.ST1 (c) in measuring RNA CHA circuit output. Indicated concentrations of gel-purified RNA hairpins H1B and H2 were incubated with equal concentration of H1BF:H1BQ or gel-purified Spinach.ST1 (+ 70 µM DFHBI) in the presence of titrating concentrations of pure C1 RNA. All circuits were operated in 1× TNaK buffer containing 20 U of RNaseOUT at 37°C, and average data from triplicate experiments are represented. Signal-to-noise ratio of H1BF:H1BQ versus Spinach.ST1 over the time course of RNA CHA detection is plotted in (d).

    Article Snippet: In vitro transcription In all, 100 pg to 1000 ng of double-stranded DNA transcription templates was transcribed using 100 U of T7 RNA polymerase (NEB) in 50 µl reactions containing 40 mM Tris–HCl, pH 7.9, 30 mM MgCl2 , 10 mM DTT, 2 mM spermidine, 4 mM ribonucleotide (rNTP) mix and 20 U of the recombinant ribonuclease inhibitor RNaseOUT (Life Technologies).

    Techniques: Polymerase Chain Reaction, Generated, Amplification, Clone Assay, Plasmid Preparation, Sequencing, Incubation, Fluorescence, Purification, Concentration Assay