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plasmids pbind, pact and pg5luc  (Promega)

 
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    Promega plasmids pbind, pact and pg5luc
    Principle and evaluation of the cell-based protease assay. (A) A protease expression construct is co-transfected with the <t>pG5luc</t> reporter plasmid into COS-1 cells. The protease construct expresses a chimeric protein which contains a GAL4 binding domain (GAL4BD) and a VP16 activation domain (VP16AD) between which part of the CVB3 polyprotein (15 C-terminal amino acids of 3A, 3B, 3C pro , and 15 N-terminal acids of 3D) is inserted. Active protease cleaves the chimeric protein at the 3C pro cleavage (arrow). If the protease is catalytically inactive, binding of GAL4BD to the GAL4 sequences in the reporter plasmid recruits VP16AD to the transcription start site, resulting in induction of FLuc expression. (B) The fusion protein is 3C pro -dependently cleaved. Plasmids pBind, pBind-VP16, pBind-3C pro (CVB3)-VP16, or pBind-3C pro [C147A](CVB3)-VP16 were co-transfected with pG5luc into COS-1 cells. The next day, cells were lysed and the proteins were separated by SDS–PAGE and stained with α-GAL4BD and α-Tubulin antibodies. (C + D) Inhibition of 3C pro activity results in induction of FLuc expression. COS-1 cells were co-transfected with protease constructs in combination with the pG5luc reporter and immediately treated with DMSO or AG7088 at 50 μM (C) or at the indicated concentrations (D). At 16 h post transfection, the cells were lysed and FLuc and RLuc were measured. Experiments were performed in triplicate and mean values ± SD are depicted.
    Plasmids Pbind, Pact And Pg5luc, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/plasmids+pbind%2C+pact+and+pg5luc/pmc07113757-62-4-13?v=Promega
    Average 90 stars, based on 1 article reviews
    plasmids pbind, pact and pg5luc - by Bioz Stars, 2026-08
    90/100 stars

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    1) Product Images from "Application of a cell-based protease assay for testing inhibitors of picornavirus 3C proteases"

    Article Title: Application of a cell-based protease assay for testing inhibitors of picornavirus 3C proteases

    Journal: Antiviral Research

    doi: 10.1016/j.antiviral.2013.12.012

    Principle and evaluation of the cell-based protease assay. (A) A protease expression construct is co-transfected with the pG5luc reporter plasmid into COS-1 cells. The protease construct expresses a chimeric protein which contains a GAL4 binding domain (GAL4BD) and a VP16 activation domain (VP16AD) between which part of the CVB3 polyprotein (15 C-terminal amino acids of 3A, 3B, 3C pro , and 15 N-terminal acids of 3D) is inserted. Active protease cleaves the chimeric protein at the 3C pro cleavage (arrow). If the protease is catalytically inactive, binding of GAL4BD to the GAL4 sequences in the reporter plasmid recruits VP16AD to the transcription start site, resulting in induction of FLuc expression. (B) The fusion protein is 3C pro -dependently cleaved. Plasmids pBind, pBind-VP16, pBind-3C pro (CVB3)-VP16, or pBind-3C pro [C147A](CVB3)-VP16 were co-transfected with pG5luc into COS-1 cells. The next day, cells were lysed and the proteins were separated by SDS–PAGE and stained with α-GAL4BD and α-Tubulin antibodies. (C + D) Inhibition of 3C pro activity results in induction of FLuc expression. COS-1 cells were co-transfected with protease constructs in combination with the pG5luc reporter and immediately treated with DMSO or AG7088 at 50 μM (C) or at the indicated concentrations (D). At 16 h post transfection, the cells were lysed and FLuc and RLuc were measured. Experiments were performed in triplicate and mean values ± SD are depicted.
    Figure Legend Snippet: Principle and evaluation of the cell-based protease assay. (A) A protease expression construct is co-transfected with the pG5luc reporter plasmid into COS-1 cells. The protease construct expresses a chimeric protein which contains a GAL4 binding domain (GAL4BD) and a VP16 activation domain (VP16AD) between which part of the CVB3 polyprotein (15 C-terminal amino acids of 3A, 3B, 3C pro , and 15 N-terminal acids of 3D) is inserted. Active protease cleaves the chimeric protein at the 3C pro cleavage (arrow). If the protease is catalytically inactive, binding of GAL4BD to the GAL4 sequences in the reporter plasmid recruits VP16AD to the transcription start site, resulting in induction of FLuc expression. (B) The fusion protein is 3C pro -dependently cleaved. Plasmids pBind, pBind-VP16, pBind-3C pro (CVB3)-VP16, or pBind-3C pro [C147A](CVB3)-VP16 were co-transfected with pG5luc into COS-1 cells. The next day, cells were lysed and the proteins were separated by SDS–PAGE and stained with α-GAL4BD and α-Tubulin antibodies. (C + D) Inhibition of 3C pro activity results in induction of FLuc expression. COS-1 cells were co-transfected with protease constructs in combination with the pG5luc reporter and immediately treated with DMSO or AG7088 at 50 μM (C) or at the indicated concentrations (D). At 16 h post transfection, the cells were lysed and FLuc and RLuc were measured. Experiments were performed in triplicate and mean values ± SD are depicted.

    Techniques Used: Protease Assay, Expressing, Construct, Transfection, Plasmid Preparation, Binding Assay, Activation Assay, SDS Page, Staining, Inhibition, Activity Assay

    Suitability of the CVB3 3C pro assay for high-throughput screening. (A) Determination of the Z ′ factor for the CVB3 3C pro assay. COS-1 cells were co-transfected with pBind-3C pro (CVB3)-VP16 and pG5luc, treated with 50 μM AG7088 and luciferase levels were measured the next day. The Gaussia curves displayed were fitted using nonlinear regression. (B) EGFP can also be used as a reporter for protease activity. COS-1 cells were co-transfected with pBind-3C pro (CVB3)-VP16 or the C147A mutant and pG5EGFP, treated with 50 μM AG7088 and EGFP was imaged after 2 days.
    Figure Legend Snippet: Suitability of the CVB3 3C pro assay for high-throughput screening. (A) Determination of the Z ′ factor for the CVB3 3C pro assay. COS-1 cells were co-transfected with pBind-3C pro (CVB3)-VP16 and pG5luc, treated with 50 μM AG7088 and luciferase levels were measured the next day. The Gaussia curves displayed were fitted using nonlinear regression. (B) EGFP can also be used as a reporter for protease activity. COS-1 cells were co-transfected with pBind-3C pro (CVB3)-VP16 or the C147A mutant and pG5EGFP, treated with 50 μM AG7088 and EGFP was imaged after 2 days.

    Techniques Used: High Throughput Screening Assay, Transfection, Luciferase, Activity Assay, Mutagenesis

    AG7088 and SG85 inhibit enterovirus 3C pro and FMDV 3C pro . (A) AG7088 and SG85 display activity against enterovirus 3C pro and FMDV 3C pro . Cells co-transfected with the constructs for the indicated proteases and pG5luc reporter were treated with 50 μM AG7088 or SG85 and the next day the luciferase levels were measured. Displayed are the FLuc levels. (B + C) AG7088 and SG85, but not AG7404 inhibit FMDV 3C pro , albeit with lower potency than CVB3 3C pro . Cells co-transfected with pG5luc and the CVB3 or FMDV 3C pro constructs were treated with a range of concentrations of AG7088 or AG7404 (B) or SG85 (C). The dashed lines represent the values obtained for the respective inactive mutants (upper dashed line) or the values obtained for the untreated wt constructs (lower dashed line). All experiments were performed in triplicate and values represent the mean FLuc ± SD.
    Figure Legend Snippet: AG7088 and SG85 inhibit enterovirus 3C pro and FMDV 3C pro . (A) AG7088 and SG85 display activity against enterovirus 3C pro and FMDV 3C pro . Cells co-transfected with the constructs for the indicated proteases and pG5luc reporter were treated with 50 μM AG7088 or SG85 and the next day the luciferase levels were measured. Displayed are the FLuc levels. (B + C) AG7088 and SG85, but not AG7404 inhibit FMDV 3C pro , albeit with lower potency than CVB3 3C pro . Cells co-transfected with pG5luc and the CVB3 or FMDV 3C pro constructs were treated with a range of concentrations of AG7088 or AG7404 (B) or SG85 (C). The dashed lines represent the values obtained for the respective inactive mutants (upper dashed line) or the values obtained for the untreated wt constructs (lower dashed line). All experiments were performed in triplicate and values represent the mean FLuc ± SD.

    Techniques Used: Activity Assay, Transfection, Construct, Luciferase



    Similar Products

    plasmids pbind, pact and pg5luc  (Promega)
    90
    Promega plasmids pbind, pact and pg5luc
    Principle and evaluation of the cell-based protease assay. (A) A protease expression construct is co-transfected with the <t>pG5luc</t> reporter plasmid into COS-1 cells. The protease construct expresses a chimeric protein which contains a GAL4 binding domain (GAL4BD) and a VP16 activation domain (VP16AD) between which part of the CVB3 polyprotein (15 C-terminal amino acids of 3A, 3B, 3C pro , and 15 N-terminal acids of 3D) is inserted. Active protease cleaves the chimeric protein at the 3C pro cleavage (arrow). If the protease is catalytically inactive, binding of GAL4BD to the GAL4 sequences in the reporter plasmid recruits VP16AD to the transcription start site, resulting in induction of FLuc expression. (B) The fusion protein is 3C pro -dependently cleaved. Plasmids pBind, pBind-VP16, pBind-3C pro (CVB3)-VP16, or pBind-3C pro [C147A](CVB3)-VP16 were co-transfected with pG5luc into COS-1 cells. The next day, cells were lysed and the proteins were separated by SDS–PAGE and stained with α-GAL4BD and α-Tubulin antibodies. (C + D) Inhibition of 3C pro activity results in induction of FLuc expression. COS-1 cells were co-transfected with protease constructs in combination with the pG5luc reporter and immediately treated with DMSO or AG7088 at 50 μM (C) or at the indicated concentrations (D). At 16 h post transfection, the cells were lysed and FLuc and RLuc were measured. Experiments were performed in triplicate and mean values ± SD are depicted.
    Plasmids Pbind, Pact And Pg5luc, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/plasmids+pbind%2C+pact+and+pg5luc/pmc07113757-62-4-13?v=Promega
    Average 90 stars, based on 1 article reviews
    plasmids pbind, pact and pg5luc - by Bioz Stars, 2026-08
    90/100 stars
    		  Buy from Supplier
    plasmids pact, pbind, and pg5luc  (Promega)
    90
    Promega plasmids pact, pbind, and pg5luc
    Principle and evaluation of the cell-based protease assay. (A) A protease expression construct is co-transfected with the <t>pG5luc</t> reporter plasmid into COS-1 cells. The protease construct expresses a chimeric protein which contains a GAL4 binding domain (GAL4BD) and a VP16 activation domain (VP16AD) between which part of the CVB3 polyprotein (15 C-terminal amino acids of 3A, 3B, 3C pro , and 15 N-terminal acids of 3D) is inserted. Active protease cleaves the chimeric protein at the 3C pro cleavage (arrow). If the protease is catalytically inactive, binding of GAL4BD to the GAL4 sequences in the reporter plasmid recruits VP16AD to the transcription start site, resulting in induction of FLuc expression. (B) The fusion protein is 3C pro -dependently cleaved. Plasmids pBind, pBind-VP16, pBind-3C pro (CVB3)-VP16, or pBind-3C pro [C147A](CVB3)-VP16 were co-transfected with pG5luc into COS-1 cells. The next day, cells were lysed and the proteins were separated by SDS–PAGE and stained with α-GAL4BD and α-Tubulin antibodies. (C + D) Inhibition of 3C pro activity results in induction of FLuc expression. COS-1 cells were co-transfected with protease constructs in combination with the pG5luc reporter and immediately treated with DMSO or AG7088 at 50 μM (C) or at the indicated concentrations (D). At 16 h post transfection, the cells were lysed and FLuc and RLuc were measured. Experiments were performed in triplicate and mean values ± SD are depicted.
    Plasmids Pact, Pbind, And Pg5luc, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/plasmids+pbind%2C+pact+and+pg5luc/10__1074_slash_jbc__m314094200-86-11-18?v=Promega
    Average 90 stars, based on 1 article reviews
    plasmids pact, pbind, and pg5luc - by Bioz Stars, 2026-08
    90/100 stars
    		  Buy from Supplier

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    Principle and evaluation of the cell-based protease assay. (A) A protease expression construct is co-transfected with the pG5luc reporter plasmid into COS-1 cells. The protease construct expresses a chimeric protein which contains a GAL4 binding domain (GAL4BD) and a VP16 activation domain (VP16AD) between which part of the CVB3 polyprotein (15 C-terminal amino acids of 3A, 3B, 3C pro , and 15 N-terminal acids of 3D) is inserted. Active protease cleaves the chimeric protein at the 3C pro cleavage (arrow). If the protease is catalytically inactive, binding of GAL4BD to the GAL4 sequences in the reporter plasmid recruits VP16AD to the transcription start site, resulting in induction of FLuc expression. (B) The fusion protein is 3C pro -dependently cleaved. Plasmids pBind, pBind-VP16, pBind-3C pro (CVB3)-VP16, or pBind-3C pro [C147A](CVB3)-VP16 were co-transfected with pG5luc into COS-1 cells. The next day, cells were lysed and the proteins were separated by SDS–PAGE and stained with α-GAL4BD and α-Tubulin antibodies. (C + D) Inhibition of 3C pro activity results in induction of FLuc expression. COS-1 cells were co-transfected with protease constructs in combination with the pG5luc reporter and immediately treated with DMSO or AG7088 at 50 μM (C) or at the indicated concentrations (D). At 16 h post transfection, the cells were lysed and FLuc and RLuc were measured. Experiments were performed in triplicate and mean values ± SD are depicted.

    Journal: Antiviral Research

    Article Title: Application of a cell-based protease assay for testing inhibitors of picornavirus 3C proteases

    doi: 10.1016/j.antiviral.2013.12.012

    Figure Lengend Snippet: Principle and evaluation of the cell-based protease assay. (A) A protease expression construct is co-transfected with the pG5luc reporter plasmid into COS-1 cells. The protease construct expresses a chimeric protein which contains a GAL4 binding domain (GAL4BD) and a VP16 activation domain (VP16AD) between which part of the CVB3 polyprotein (15 C-terminal amino acids of 3A, 3B, 3C pro , and 15 N-terminal acids of 3D) is inserted. Active protease cleaves the chimeric protein at the 3C pro cleavage (arrow). If the protease is catalytically inactive, binding of GAL4BD to the GAL4 sequences in the reporter plasmid recruits VP16AD to the transcription start site, resulting in induction of FLuc expression. (B) The fusion protein is 3C pro -dependently cleaved. Plasmids pBind, pBind-VP16, pBind-3C pro (CVB3)-VP16, or pBind-3C pro [C147A](CVB3)-VP16 were co-transfected with pG5luc into COS-1 cells. The next day, cells were lysed and the proteins were separated by SDS–PAGE and stained with α-GAL4BD and α-Tubulin antibodies. (C + D) Inhibition of 3C pro activity results in induction of FLuc expression. COS-1 cells were co-transfected with protease constructs in combination with the pG5luc reporter and immediately treated with DMSO or AG7088 at 50 μM (C) or at the indicated concentrations (D). At 16 h post transfection, the cells were lysed and FLuc and RLuc were measured. Experiments were performed in triplicate and mean values ± SD are depicted.

    Article Snippet: Plasmids pBind, pAct and pG5luc were derived from the CheckMateTM Mammalian Two-Hybrid System (Promega). pBind-VP16 was produced by ligating the VP16AD-coding sequence amplified from pAct into the XbaI and NotI sites of the multiple cloning site of pBind. pBind-VP16 was subsequently used for cloning all protease constructs using the SalI and MluI sites between GAL4BD and VP16AD.

    Techniques: Protease Assay, Expressing, Construct, Transfection, Plasmid Preparation, Binding Assay, Activation Assay, SDS Page, Staining, Inhibition, Activity Assay

    Suitability of the CVB3 3C pro assay for high-throughput screening. (A) Determination of the Z ′ factor for the CVB3 3C pro assay. COS-1 cells were co-transfected with pBind-3C pro (CVB3)-VP16 and pG5luc, treated with 50 μM AG7088 and luciferase levels were measured the next day. The Gaussia curves displayed were fitted using nonlinear regression. (B) EGFP can also be used as a reporter for protease activity. COS-1 cells were co-transfected with pBind-3C pro (CVB3)-VP16 or the C147A mutant and pG5EGFP, treated with 50 μM AG7088 and EGFP was imaged after 2 days.

    Journal: Antiviral Research

    Article Title: Application of a cell-based protease assay for testing inhibitors of picornavirus 3C proteases

    doi: 10.1016/j.antiviral.2013.12.012

    Figure Lengend Snippet: Suitability of the CVB3 3C pro assay for high-throughput screening. (A) Determination of the Z ′ factor for the CVB3 3C pro assay. COS-1 cells were co-transfected with pBind-3C pro (CVB3)-VP16 and pG5luc, treated with 50 μM AG7088 and luciferase levels were measured the next day. The Gaussia curves displayed were fitted using nonlinear regression. (B) EGFP can also be used as a reporter for protease activity. COS-1 cells were co-transfected with pBind-3C pro (CVB3)-VP16 or the C147A mutant and pG5EGFP, treated with 50 μM AG7088 and EGFP was imaged after 2 days.

    Article Snippet: Plasmids pBind, pAct and pG5luc were derived from the CheckMateTM Mammalian Two-Hybrid System (Promega). pBind-VP16 was produced by ligating the VP16AD-coding sequence amplified from pAct into the XbaI and NotI sites of the multiple cloning site of pBind. pBind-VP16 was subsequently used for cloning all protease constructs using the SalI and MluI sites between GAL4BD and VP16AD.

    Techniques: High Throughput Screening Assay, Transfection, Luciferase, Activity Assay, Mutagenesis

    AG7088 and SG85 inhibit enterovirus 3C pro and FMDV 3C pro . (A) AG7088 and SG85 display activity against enterovirus 3C pro and FMDV 3C pro . Cells co-transfected with the constructs for the indicated proteases and pG5luc reporter were treated with 50 μM AG7088 or SG85 and the next day the luciferase levels were measured. Displayed are the FLuc levels. (B + C) AG7088 and SG85, but not AG7404 inhibit FMDV 3C pro , albeit with lower potency than CVB3 3C pro . Cells co-transfected with pG5luc and the CVB3 or FMDV 3C pro constructs were treated with a range of concentrations of AG7088 or AG7404 (B) or SG85 (C). The dashed lines represent the values obtained for the respective inactive mutants (upper dashed line) or the values obtained for the untreated wt constructs (lower dashed line). All experiments were performed in triplicate and values represent the mean FLuc ± SD.

    Journal: Antiviral Research

    Article Title: Application of a cell-based protease assay for testing inhibitors of picornavirus 3C proteases

    doi: 10.1016/j.antiviral.2013.12.012

    Figure Lengend Snippet: AG7088 and SG85 inhibit enterovirus 3C pro and FMDV 3C pro . (A) AG7088 and SG85 display activity against enterovirus 3C pro and FMDV 3C pro . Cells co-transfected with the constructs for the indicated proteases and pG5luc reporter were treated with 50 μM AG7088 or SG85 and the next day the luciferase levels were measured. Displayed are the FLuc levels. (B + C) AG7088 and SG85, but not AG7404 inhibit FMDV 3C pro , albeit with lower potency than CVB3 3C pro . Cells co-transfected with pG5luc and the CVB3 or FMDV 3C pro constructs were treated with a range of concentrations of AG7088 or AG7404 (B) or SG85 (C). The dashed lines represent the values obtained for the respective inactive mutants (upper dashed line) or the values obtained for the untreated wt constructs (lower dashed line). All experiments were performed in triplicate and values represent the mean FLuc ± SD.

    Article Snippet: Plasmids pBind, pAct and pG5luc were derived from the CheckMateTM Mammalian Two-Hybrid System (Promega). pBind-VP16 was produced by ligating the VP16AD-coding sequence amplified from pAct into the XbaI and NotI sites of the multiple cloning site of pBind. pBind-VP16 was subsequently used for cloning all protease constructs using the SalI and MluI sites between GAL4BD and VP16AD.

    Techniques: Activity Assay, Transfection, Construct, Luciferase