TREX1 exonuclease activity was assayed as described in Hasan et al. (2015) (link). Briefly, approximately 1 × 106 293T were transfected with either WT or mutant TREX1-V5 plasmids. Cells were lysed in IP buffer (20 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.5% NP-40, and 1× protease inhibitor), and the post-nuclear supernatant was isolated by centrifugation at 20,000 × g and mixed with anti-V5 agarose beads (Bethyl Labs). Upon overnight incubation at 4°C, the beads were subsequently washed twice with IP buffer and twice with low-salt IP buffer (50 mM NaCl). Washed beads were resuspended in 50 μL of DNase buffer (20 mM Tris-HCl [pH 7.4], 5 mM MgCl2, 2 mM DTT, 100 mg/mL BSA, and 0.5% NP-40). Bead-bound TREX1 was subject to either in vitro phosphorylation or DNase assay. For TREX1 in vitro phosphorylation, beads bound TREX1 was incubated with 20U of recombinant CDK1 (NEB) following the manufacturer’s suggestion. Samples were later analyzed by Phos-tag immunoblotting. To assess TREX1 DNase function, samples were divided into three equal volumes and mixed with 90 μL of pre-warmed to 37°C DNase reaction buffer (20 mM Tris-HCl [pH 7.4], 5 mM MgCl2, 2 mM DTT, 100 mg/mL BSA, 1/1,200 SYBR Green, and 5 ng/μl or 10 ng/μl ssDNA) followed by real-time quantification of the DNA/SYBR complex using a SynergyHT microplate reader (Biotek).
Recombinant cdk1
Manufactured by New England Biolabs
Recombinant CDK1 is a purified, recombinant protein that represents the catalytic subunit of the Cyclin-Dependent Kinase 1 (CDK1) enzyme. CDK1 is a key regulator of the cell cycle and plays a crucial role in the progression from the G2 phase to the M phase of the cell cycle.
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Lab products found in correlation
2 protocols using recombinant cdk1
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TREX1 Exonuclease Activity Assay
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TREX1 Exonuclease Activity Assay
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TREX1 exonuclease activity was assayed as described in Hasan et al. (2015) (link). Briefly, approximately 1 × 106 293T were transfected with either WT or mutant TREX1-V5 plasmids. Cells were lysed in IP buffer (20 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.5% NP-40, and 1× protease inhibitor), and the post-nuclear supernatant was isolated by centrifugation at 20,000 × g and mixed with anti-V5 agarose beads (Bethyl Labs). Upon overnight incubation at 4°C, the beads were subsequently washed twice with IP buffer and twice with low-salt IP buffer (50 mM NaCl). Washed beads were resuspended in 50 μL of DNase buffer (20 mM Tris-HCl [pH 7.4], 5 mM MgCl2, 2 mM DTT, 100 mg/mL BSA, and 0.5% NP-40). Bead-bound TREX1 was subject to either in vitro phosphorylation or DNase assay. For TREX1 in vitro phosphorylation, beads bound TREX1 was incubated with 20U of recombinant CDK1 (NEB) following the manufacturer’s suggestion. Samples were later analyzed by Phos-tag immunoblotting. To assess TREX1 DNase function, samples were divided into three equal volumes and mixed with 90 μL of pre-warmed to 37°C DNase reaction buffer (20 mM Tris-HCl [pH 7.4], 5 mM MgCl2, 2 mM DTT, 100 mg/mL BSA, 1/1,200 SYBR Green, and 5 ng/μl or 10 ng/μl ssDNA) followed by real-time quantification of the DNA/SYBR complex using a SynergyHT microplate reader (Biotek).
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