Neg502a100uc

Manufactured by PerkinElmer

The NEG502A100UC is a laboratory instrument manufactured by PerkinElmer. It is designed for the purpose of performing specific laboratory functions, though a detailed description is not available while maintaining an unbiased and factual approach.

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Lab products found in correlation

Instantblue protein stain, by Abcam (1 mentions) Srp5096, by Merck Group (1 mentions) Photographic film, by Bio-Rad (1 mentions) Nupage acrylamide gel, by Thermo Fisher Scientific (1 mentions) Vent dna polymerase, by New England Biolabs (1 mentions) 14c leucine, by PerkinElmer (1 mentions) Nec279e001mc, by PerkinElmer (1 mentions) Sp6 rna polymerase, by Thermo Fisher Scientific (1 mentions) 3h leucine, by PerkinElmer (1 mentions) Dna purification kit, by Zymo Research (1 mentions) γ 32p atp, by PerkinElmer (1 mentions) Restriction endonuclease, by New England Biolabs (1 mentions) Quick ligase, by New England Biolabs (1 mentions)

2 protocols using neg502a100uc

In vitro Radioactive Kinase Assay

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In vitro radioactive assays were performed by incubating 100 ng recombinant ATG4B diluted in assay buffer (20 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 5 mM DTT, 20 µM cold ATP and 0.16 µM ATP [γ-32P] PerkinElmer NEG502A100UC) in the presence of 10 ng recombinant ULK1 (catalytic domain) or 3 µL of active recombinant ULK1 (Sigma-Aldrich, SRP5096) at 30 °C for the indicated times. The reaction was stopped by adding 5× sample buffer (250 mM Tris-Cl pH 6.8, 10% sodium dodecyl sulphate (SDS), 50% glycerol, 25% β-mercaptoethanol or 0.5 M DTT and 0.05% bromophenol blue) and boiling for 5 min. Samples were loaded on NUPAGE Acrylamide gel (Invitrogen, NP0321BOX). Gels were stained with InstantBlue Protein Stain (Expedeon, ISB1L) before drying on filter paper and measuring incorporated radioactivity by exposing on photographic film (Bio-Rad).

Pengo N., Agrotis A., Prak K., Jones J, & Ketteler R. (2017). A reversible phospho-switch mediated by ULK1 regulates the activity of autophagy protease ATG4B. Nature Communications, 8, 294.

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Radiolabeled Reagents for Molecular Biology

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[γ−³²P]ATP, [¹⁴C]leucine, and [³H]leucine (catalogue numbers NEG502A100UC, NEC279E001MC, and NET1166001MC, respectively) were from Perkin-Elmer (Waltham, MA). Restriction endonucleases, Vent DNA polymerase, Quick ligase, and all DNA modifying enzymes were from New England Biolabs (Ipswich, MA) (catalogue numbers: Bam HI-HF, R3136; Pst I, R0140, Vent DNA polymerase, M0254; Quick ligase, M2200; Hind III-HF, R3104). Amino-acid-free rabbit reticulocyte lysate was purchased in bulk, custom-made by Ambion (Austin, TX), SP6 RNA polymerase was prepared as described (Gurevich 1996 (link)). DNA purification kits were from Zymo Research (Irvine, CA). All other reagents were from Sigma-Aldrich (St. Louis, MO) or Amresco (Cleveland, OH), as indicated.

Vishnivetskiy S.A., Huh E.K., Gurevich E.V, & Gurevich V.V. (2020). The finger loop as an activation sensor in arrestin.. Journal of neurochemistry, 157(4), 1138-1152.

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