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macrogreen expression plasmid  (Addgene inc)


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    Addgene inc macrogreen expression plasmid
    Comparison between <t>MacroGreen</t> and the wild type Af1521-GFP fusion binding to either PARylated PARP1 ( a ) or MARylated PARP10 ( b ) coated plates. In total, 50 µl reactions in 96-well Nunc MaxiSorp TM plates; detection with 1 µM MacroGreen or Af1521-GFP at room temperature c Examples of the linear assay range using MacroGreen detection of PARP1 PARylation predominantly at acidic side chains; PARP1 PARylation predominantly at serine side chains in the presence of HPF1; PARP10 MARylation at various side chains; or C2I MARylation of actin at Arg177. d Extended linear range of MAR detection on PARP10 under the same conditions. e Detection of MAR on five histone proteins (H2B, H3.1, H3.2, H3.3 and H4 as indicated) in the presence of PARP10. PARP10 MARylation alone (gray) serves as reference. Negative control reactions contained NAD + (1 mM) but not PARP10. Statistical significance of the data was calculated using a one-way ANOVA test; error bars indicate S.D. All panels – n = 4.
    Macrogreen Expression Plasmid, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/expression+vector+plpcx/pLPC-NMYC+TRF2+(Plasmid+%2316066)/pmc08319303-159-1-7
    Average 93 stars, based on 15 article reviews
    macrogreen expression plasmid - by Bioz Stars, 2026-11
    93/100 stars

    Images

    1) Product Images from "MacroGreen, a simple tool for detection of ADP-ribosylated proteins"

    Article Title: MacroGreen, a simple tool for detection of ADP-ribosylated proteins

    Journal: Communications Biology

    doi: 10.1038/s42003-021-02439-w

    Comparison between MacroGreen and the wild type Af1521-GFP fusion binding to either PARylated PARP1 ( a ) or MARylated PARP10 ( b ) coated plates. In total, 50 µl reactions in 96-well Nunc MaxiSorp TM plates; detection with 1 µM MacroGreen or Af1521-GFP at room temperature c Examples of the linear assay range using MacroGreen detection of PARP1 PARylation predominantly at acidic side chains; PARP1 PARylation predominantly at serine side chains in the presence of HPF1; PARP10 MARylation at various side chains; or C2I MARylation of actin at Arg177. d Extended linear range of MAR detection on PARP10 under the same conditions. e Detection of MAR on five histone proteins (H2B, H3.1, H3.2, H3.3 and H4 as indicated) in the presence of PARP10. PARP10 MARylation alone (gray) serves as reference. Negative control reactions contained NAD + (1 mM) but not PARP10. Statistical significance of the data was calculated using a one-way ANOVA test; error bars indicate S.D. All panels – n = 4.
    Figure Legend Snippet: Comparison between MacroGreen and the wild type Af1521-GFP fusion binding to either PARylated PARP1 ( a ) or MARylated PARP10 ( b ) coated plates. In total, 50 µl reactions in 96-well Nunc MaxiSorp TM plates; detection with 1 µM MacroGreen or Af1521-GFP at room temperature c Examples of the linear assay range using MacroGreen detection of PARP1 PARylation predominantly at acidic side chains; PARP1 PARylation predominantly at serine side chains in the presence of HPF1; PARP10 MARylation at various side chains; or C2I MARylation of actin at Arg177. d Extended linear range of MAR detection on PARP10 under the same conditions. e Detection of MAR on five histone proteins (H2B, H3.1, H3.2, H3.3 and H4 as indicated) in the presence of PARP10. PARP10 MARylation alone (gray) serves as reference. Negative control reactions contained NAD + (1 mM) but not PARP10. Statistical significance of the data was calculated using a one-way ANOVA test; error bars indicate S.D. All panels – n = 4.

    Techniques Used: Comparison, Binding Assay, Negative Control

    a Verification of PARP1 inhibition by Talazoparib and of PARP10 inhibition by PJ-34 using Western blotting. b Quantification using MacroGreen of PARP1 and PARP10 inhibition. n = 4; error bars indicate S.D.
    Figure Legend Snippet: a Verification of PARP1 inhibition by Talazoparib and of PARP10 inhibition by PJ-34 using Western blotting. b Quantification using MacroGreen of PARP1 and PARP10 inhibition. n = 4; error bars indicate S.D.

    Techniques Used: Inhibition, Western Blot

    a MacroGreen staining of DNA damage sites in U2OS DNA double-strand break reporter cells, verified by staining for γH2AX. Control experiments contained a PARP1 inhibitor. Scale bars: Main panel, 10 μm; inset, 2.5 μm. b MacroGreen fluorescence detected after staining of HEK293T cells grown in 96-well plates and treated to induce DNA damage as indicated. n = 12; error bars indicate S.D.
    Figure Legend Snippet: a MacroGreen staining of DNA damage sites in U2OS DNA double-strand break reporter cells, verified by staining for γH2AX. Control experiments contained a PARP1 inhibitor. Scale bars: Main panel, 10 μm; inset, 2.5 μm. b MacroGreen fluorescence detected after staining of HEK293T cells grown in 96-well plates and treated to induce DNA damage as indicated. n = 12; error bars indicate S.D.

    Techniques Used: Staining, Control, Fluorescence

    Related Articles

    Sequencing:

    Article Title: Phosphorylation of TRF2 promotes its interaction with TIN2 and regulates DNA damage response at telomeres.
    Article Snippet: Expression of Cas9 was induced in iCut-RPE1 cells by overnight treatment with doxy cy cline and Shield-1 (1 M, Aobious) and telomeric DNA damage was generated by transfection of the synthetic sgRNA TT AGGGTT AGGGTT AGGGTT (Sigma) as described previously ( 46 , 47 ). sgRNA was transfected by Lipofectamine RNAiMAX (Thermofisher) at final concentration 5 nM. .. Coding sequence of human TRF2 was PCR amplified from pLPC-NMyc-TRF2 (Addgene ID: 16066) ( 48 ) and inserted in frame into pEGFP plasmid. .. Mutagenesis of TRF2 was performed using PCR amplification followed by ligation of DNA fragments into pEGFP backbone by Gibson assembly kit (NEB).

    Polymerase Chain Reaction:

    Article Title: Phosphorylation of TRF2 promotes its interaction with TIN2 and regulates DNA damage response at telomeres.
    Article Snippet: Expression of Cas9 was induced in iCut-RPE1 cells by overnight treatment with doxy cy cline and Shield-1 (1 M, Aobious) and telomeric DNA damage was generated by transfection of the synthetic sgRNA TT AGGGTT AGGGTT AGGGTT (Sigma) as described previously ( 46 , 47 ). sgRNA was transfected by Lipofectamine RNAiMAX (Thermofisher) at final concentration 5 nM. .. Coding sequence of human TRF2 was PCR amplified from pLPC-NMyc-TRF2 (Addgene ID: 16066) ( 48 ) and inserted in frame into pEGFP plasmid. .. Mutagenesis of TRF2 was performed using PCR amplification followed by ligation of DNA fragments into pEGFP backbone by Gibson assembly kit (NEB).

    Article Title: A CPC-shelterin-BTR axis regulates mitotic telomere deprotection
    Article Snippet: .. Flag and myc tags were added during PCR. pLPC-Myc and pLPC-Myc-TRF2 expressing the short TRF2 isoform were acquired from Addgene (Plasmids #12540 and #16066 respectively, kind gift of Tita de Lange). .. Mutations and deletions were introduced by site-directed PCR mutagenesis (TRF1, TRF2, BLM) or during artificial DNA synthesis (TOP3A) and cloned by the recombination-based HiFi assembly strategy (NEB). mScarlet and mClover/mClover3 were in-frame cloned at 5’-terminus of 3FL-TRF1 or TRF2 genes with (GGGGS)x2 linker sequence.

    Amplification:

    Article Title: Phosphorylation of TRF2 promotes its interaction with TIN2 and regulates DNA damage response at telomeres.
    Article Snippet: Expression of Cas9 was induced in iCut-RPE1 cells by overnight treatment with doxy cy cline and Shield-1 (1 M, Aobious) and telomeric DNA damage was generated by transfection of the synthetic sgRNA TT AGGGTT AGGGTT AGGGTT (Sigma) as described previously ( 46 , 47 ). sgRNA was transfected by Lipofectamine RNAiMAX (Thermofisher) at final concentration 5 nM. .. Coding sequence of human TRF2 was PCR amplified from pLPC-NMyc-TRF2 (Addgene ID: 16066) ( 48 ) and inserted in frame into pEGFP plasmid. .. Mutagenesis of TRF2 was performed using PCR amplification followed by ligation of DNA fragments into pEGFP backbone by Gibson assembly kit (NEB).

    Article Title: Essential gene knockdowns reveal genetic vulnerabilities and antibiotic sensitivities in Acinetobacter baumannii .
    Article Snippet: .. The Mobile-CRISPRi plasmid pJMP2748 is a variant of pJMP2754 (Addgene 160666) with the sgRNA promoter derived from pJMP2367 (Addgene 160076) and the dCas9 promoter region amplified from the A. baumannii suppressor strain gDNA with oJMP635 and oJMP636. ..

    Article Title: Essential gene knockdowns reveal genetic vulnerabilities and antibiotic sensitivities in Acinetobacter baumannii
    Article Snippet: .. The Mobile-CRISPRi plasmid pJMP2748 is a variant of pJMP2754 (Addgene 160666) with the sgRNA promoter derived from pJMP2367 (Addgene 160076) and the dCas9 promoter region amplified from the A. baumannii suppressor strain gDNA with oJMP635 and oJMP636. ..

    Article Title: Essential Gene Knockdowns Reveal Genetic Vulnerabilities and Antibiotic Sensitivities in Acinetobacter baumannii
    Article Snippet: .. The Mobile-CRISPRi plasmid pJMP2748 is a variant of pJMP2754 (Addgene 160666) with the sgRNA promoter derived from pJMP2367 (Addgene 160076) and the dCas9 promoter region amplified from the A. baumannii suppressor strain gDNA with oJMP635 and oJMP636. ..

    Plasmid Preparation:

    Article Title: Phosphorylation of TRF2 promotes its interaction with TIN2 and regulates DNA damage response at telomeres.
    Article Snippet: Expression of Cas9 was induced in iCut-RPE1 cells by overnight treatment with doxy cy cline and Shield-1 (1 M, Aobious) and telomeric DNA damage was generated by transfection of the synthetic sgRNA TT AGGGTT AGGGTT AGGGTT (Sigma) as described previously ( 46 , 47 ). sgRNA was transfected by Lipofectamine RNAiMAX (Thermofisher) at final concentration 5 nM. .. Coding sequence of human TRF2 was PCR amplified from pLPC-NMyc-TRF2 (Addgene ID: 16066) ( 48 ) and inserted in frame into pEGFP plasmid. .. Mutagenesis of TRF2 was performed using PCR amplification followed by ligation of DNA fragments into pEGFP backbone by Gibson assembly kit (NEB).

    Article Title: Essential gene knockdowns reveal genetic vulnerabilities and antibiotic sensitivities in Acinetobacter baumannii .
    Article Snippet: .. The Mobile-CRISPRi plasmid pJMP2748 is a variant of pJMP2754 (Addgene 160666) with the sgRNA promoter derived from pJMP2367 (Addgene 160076) and the dCas9 promoter region amplified from the A. baumannii suppressor strain gDNA with oJMP635 and oJMP636. ..

    Article Title: Essential gene knockdowns reveal genetic vulnerabilities and antibiotic sensitivities in Acinetobacter baumannii
    Article Snippet: .. The Mobile-CRISPRi plasmid pJMP2748 is a variant of pJMP2754 (Addgene 160666) with the sgRNA promoter derived from pJMP2367 (Addgene 160076) and the dCas9 promoter region amplified from the A. baumannii suppressor strain gDNA with oJMP635 and oJMP636. ..

    Article Title: Essential Gene Knockdowns Reveal Genetic Vulnerabilities and Antibiotic Sensitivities in Acinetobacter baumannii
    Article Snippet: .. The Mobile-CRISPRi plasmid pJMP2748 is a variant of pJMP2754 (Addgene 160666) with the sgRNA promoter derived from pJMP2367 (Addgene 160076) and the dCas9 promoter region amplified from the A. baumannii suppressor strain gDNA with oJMP635 and oJMP636. ..

    Expressing:

    Article Title: A CPC-shelterin-BTR axis regulates mitotic telomere deprotection
    Article Snippet: .. Flag and myc tags were added during PCR. pLPC-Myc and pLPC-Myc-TRF2 expressing the short TRF2 isoform were acquired from Addgene (Plasmids #12540 and #16066 respectively, kind gift of Tita de Lange). .. Mutations and deletions were introduced by site-directed PCR mutagenesis (TRF1, TRF2, BLM) or during artificial DNA synthesis (TOP3A) and cloned by the recombination-based HiFi assembly strategy (NEB). mScarlet and mClover/mClover3 were in-frame cloned at 5’-terminus of 3FL-TRF1 or TRF2 genes with (GGGGS)x2 linker sequence.

    other:

    Article Title: Phosphorylation of TRF2 promotes its interaction with TIN2 and regulates DNA damage response at telomeres
    Article Snippet: sgRNA was transfected by Lipofectamine RNAiMAX (Thermofisher) at final concentration 5 nM.

    Variant Assay:

    Article Title: Essential gene knockdowns reveal genetic vulnerabilities and antibiotic sensitivities in Acinetobacter baumannii .
    Article Snippet: .. The Mobile-CRISPRi plasmid pJMP2748 is a variant of pJMP2754 (Addgene 160666) with the sgRNA promoter derived from pJMP2367 (Addgene 160076) and the dCas9 promoter region amplified from the A. baumannii suppressor strain gDNA with oJMP635 and oJMP636. ..

    Article Title: Essential gene knockdowns reveal genetic vulnerabilities and antibiotic sensitivities in Acinetobacter baumannii
    Article Snippet: .. The Mobile-CRISPRi plasmid pJMP2748 is a variant of pJMP2754 (Addgene 160666) with the sgRNA promoter derived from pJMP2367 (Addgene 160076) and the dCas9 promoter region amplified from the A. baumannii suppressor strain gDNA with oJMP635 and oJMP636. ..

    Article Title: Essential Gene Knockdowns Reveal Genetic Vulnerabilities and Antibiotic Sensitivities in Acinetobacter baumannii
    Article Snippet: .. The Mobile-CRISPRi plasmid pJMP2748 is a variant of pJMP2754 (Addgene 160666) with the sgRNA promoter derived from pJMP2367 (Addgene 160076) and the dCas9 promoter region amplified from the A. baumannii suppressor strain gDNA with oJMP635 and oJMP636. ..

    Derivative Assay:

    Article Title: Essential gene knockdowns reveal genetic vulnerabilities and antibiotic sensitivities in Acinetobacter baumannii .
    Article Snippet: .. The Mobile-CRISPRi plasmid pJMP2748 is a variant of pJMP2754 (Addgene 160666) with the sgRNA promoter derived from pJMP2367 (Addgene 160076) and the dCas9 promoter region amplified from the A. baumannii suppressor strain gDNA with oJMP635 and oJMP636. ..

    Article Title: Essential gene knockdowns reveal genetic vulnerabilities and antibiotic sensitivities in Acinetobacter baumannii
    Article Snippet: .. The Mobile-CRISPRi plasmid pJMP2748 is a variant of pJMP2754 (Addgene 160666) with the sgRNA promoter derived from pJMP2367 (Addgene 160076) and the dCas9 promoter region amplified from the A. baumannii suppressor strain gDNA with oJMP635 and oJMP636. ..

    Article Title: Essential Gene Knockdowns Reveal Genetic Vulnerabilities and Antibiotic Sensitivities in Acinetobacter baumannii
    Article Snippet: .. The Mobile-CRISPRi plasmid pJMP2748 is a variant of pJMP2754 (Addgene 160666) with the sgRNA promoter derived from pJMP2367 (Addgene 160076) and the dCas9 promoter region amplified from the A. baumannii suppressor strain gDNA with oJMP635 and oJMP636. ..



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