Activated dna

Manufactured by BPS Biosciences

Sourced in United States

Activated DNA is a laboratory product that has been chemically modified to improve its reactivity and facilitate various experimental applications. It provides a convenient source of DNA material for downstream processes without the need for further preparation.

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Lab products found in correlation

Nupage novex 4 12 bis tris gel, by Thermo Fisher Scientific (3 mentions) Mops buffer, by Thermo Fisher Scientific (3 mentions) Spintrap g 25, by GE Healthcare (2 mentions) Mgcl2, by Merck Group (2 mentions) Anti gst antibody, by GenScript (1 mentions) Anti his antibody, by Thermo Fisher Scientific (1 mentions) Anti his antibody, by GenScript (1 mentions) Streptavidin hrp, by Thermo Fisher Scientific (1 mentions) Parp1 hsa, by Bio-Techne (1 mentions) Recombinant parp1 protein, by Bio-Techne (1 mentions)

5 protocols using activated dna

Enzymatic Hydrolysis of PARylated and MARylated PARP1

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PARylated and MARylated PARP1 proteins were prepared as described [26 (link)] in a reaction buffer containing 50 mM Tris-HCl (pH 8.0), 4 mM MgCl₂, 50 mM NaCl, 0.2 mM DTT, 200 μM NAD+ (Trevigen) and 130 ng activated DNA (BPS Bioscience). Briefly, for the PAR hydrolysis activity assays, 70 nM PARP1 (PARP1-HSA; Trevigen) was auto-modified as described in [26 (link)]. After 20 min incubation, of PARP1 was passed three times through SpinTrap G-25 (GE Healthcare). PARylated PARP1 substrate was used in 10 μL reaction. For the MARylated PARP1, 1 μM of PARP1-E988Q was used as a substrate. Reactions were stopped by the addition of PARP inhibitor Olaparib (1 μM). The MgCl₂ (Sigma) concentration was adjusted to 15 mM to allow full hydrolase activity. Automodified PARP1 was then incubated for indicated times at 30°C with hydrolytic enzymes in 10 μL reaction. Concentrations of hydrolytic enzymes used are as indicated in figures. Reactions were stopped by addition of Laemmli loading buffer, samples boiled at 90°C for 1.5 minutes and analysed by NuPAGE Novex Bis-Tris 4–12% Gel using MOPS buffer (Invitrogen). Radiolabelled experiments were visualized by autoradiography.

Palazzo L., Daniels C.M., Nettleship J.E., Rahman N., McPherson R.L., Ong S., Kato K., Nureki O., Leung A.K, & Ahel I. (2016). ENPP1 processes protein ADP‐ribosylation in vitro. The Febs Journal, 283(18), 3371-3388.

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In vitro PARylation and Phosphorylation Assays

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In vitro PARylation assays were performed as described previously (Kong et al., 2021 (link); Yao et al., 2021 (link)). Briefly, 500 ng HIS-FonPARP1 were incubated in a 50 μL PARylation buffer (50 mM Tris–HCl, 50 mM NaCl, 10 mM MgCl₂, pH8.0) with 0.2 mM NAD⁺, 1× activated DNA (BPS Bioscience, San Diego, CA, USA), and 3 μg GST-FonKin4 at 26°C for 3 h. Subsequently, the samples were separated on 8% and 12.5% SDS-PAGE. PARylated proteins were detected via immunoblotting with anti-poly-ADPR antibody (Cat. #MABE1031, Sigma-Aldrich), anti-GST antibody (Cat. #A00865, GenScript) or anti-HIS antibody (Cat. #A00186, GenScript).
For in vitro phosphorylation-mediated self-PARylation assays, 4 μg HIS-FonPARP1 underwent prior in vitro phosphorylation by HIS-FonKin4-ST, as described above. Then, 50 μL PARylation buffer was then added, followed by incubation at 26°C for 30 min. The reaction was stopped by adding 4× SDS loading buffer, and the proteins were then separated on 8% and 12.5% SDS-PAGE. Phosphorylated proteins were detected by anti-phosphor Ser/Thr antibody. PARylated proteins detected through immunoblotting with streptavidin-HRP (Cat. #21126, Thermo Fisher Scientific, Waltham, MA, USA) for Biotin NAD⁺ or anti-HIS antibody (Cat. #A00186, GenScript).

Wang J., Gao Y., Xiong X., Yan Y., Lou J., Noman M., Li D, & Song F. (2024). The Ser/Thr protein kinase FonKin4-poly(ADP-ribose) polymerase FonPARP1 phosphorylation cascade is required for the pathogenicity of watermelon fusarium wilt fungus Fusarium oxysporum f. sp. niveum. Frontiers in Microbiology, 15, 1397688.

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PARP1 Hydrolysis and Automodification Assays

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PARylated and MARylated PARP1 proteins were prepared as described 26 in a reaction buffer containing 50 mm Tris/HCl (pH 8.0), 4 mm MgCl₂, 50 mm NaCl, 0.2 mm DTT, 200 μm NAD+ (Trevigen) and 130 ng activated DNA (BPS Bioscience, Inc., San Diego, CA, USA). Briefly, for the PAR hydrolysis activity assays, 70 nm PARP1 (PARP1‐HSA; Trevigen) was automodified as described in 26. After 20‐min incubation, PARP1 was passed three times through SpinTrap G‐25 (GE Healthcare). PARylated PARP1 substrate was used in a 10‐μL reaction. For the MARylated PARP1, 1 μm of PARP1‐E988Q was used as a substrate. Reactions were stopped by the addition of PARP inhibitor Olaparib (1 μm). The MgCl₂ (Sigma) concentration was adjusted to 15 mm to allow full hydrolase activity. Automodified PARP1 was then incubated for indicated times at 30 °C with hydrolytic enzymes in 10‐μL reaction. Concentrations of hydrolytic enzymes used are as indicated in figures. Reactions were stopped by addition of Laemmli loading buffer, samples boiled at 90 °C for 1.5 min and analysed by NuPAGE Novex Bis‐Tris 4–12% gel using MOPS buffer (Invitrogen). Radiolabelled experiments were visualized by autoradiography.

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Preparation and Hydrolysis of PARylated and MARylated PARP1

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Poly(ADP-ribosyl)ated and mono(ADP-ribosylated) PARP1 proteins were prepared as described [15 (link),29 (link)] in a reaction buffer containing 50 mM Tris-HCl (pH 8.0), 4 mM MgCl₂, 50 mM NaCl, 0.2 mM DTT, 200 mM NAD⁺ (Trevigen) and 130 ng activated DNA (BPS Bioscience). For the PAR hydrolysis activity assays 0.5 μM PARylated PARP1 substrate was used in 10 μL reaction. We estimate that the average number of the ADP-ribose units in chains linked to a PARP1 protein prepared this way is 40 units per protein, giving an approximate concentration of 20 µM monomeric units in the reaction. For the MARylated PARP1, 2 μM of PARP1-E988Q was used as a substrate. Reactions were stopped by the addition of PARP1 inhibitor Olaparib (1 μM). The MgCl₂ (Sigma) concentration was adjusted to 15 mM to allow full Nudix hydrolase activity. Automodified PARP1 was then incubated for 3 hours at 30°C with hydrolytic enzymes in 10 μL reaction. Concentrations of hydrolytic enzymes used are as indicated in figures. Reactions were stopped by addition of Laemmli loading buffer, samples boiled at 90°C for 1.5 minutes and fractionated by NuPAGE Novex Bis-Tris 4-12% Gel using MOPS buffer (Invitrogen). PARP1-E988Q experiments were visualized by autoradiography, experiments using wild-type PARP1 were visualized by Western blot using a specific anti-PAR antibody.

Palazzo L., Thomas B., Jemth A.S., Colby T., Leidecker O., Feijs K., Zaja R., Loseva O., Puigvert J.C., Matic I., Helleday T, & Ahel I. (2015). Processing of protein ADP-ribosylation by Nudix hydrolases. The Biochemical journal, 468(2), 293-301.

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In vitro PARylation Assay of GST-MIF

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In vitro PARylation assay was conducted as previously described with some modifications^{61 (link)}. Briefly, GST and GST-MIF (500 ng) bound to GSH beads were incubated with 1 U recombinant PARP1 protein (Trevigen) in the reaction buffer containing 50 mM Tris-HCl (pH 8.0), 20 mM NaCl, 10 mM MgCl₂, 1 mM DTT, 1× activated DNA (BPS Bioscience) and 500 µM NAD⁺ at 37 °C for 30 min. The reaction was terminated by collecting supernatant and beads separately with the addition of SDS loading buffer. PARylation of interested proteins was detected by immunoblot assay with an anti-PAR antibody.

Wang Y., Chen Y., Wang C., Yang M., Wang Y., Bao L., Wang J.E., Kim B., Chan K.Y., Xu W., Capota E., Ortega J., Nijhawan D., Li G.M., Luo W, & Wang Y. (2021). MIF is a 3’ flap nuclease that facilitates DNA replication and promotes tumor growth. Nature Communications, 12, 2954.

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