Radioactive γ 32p atp

MG132(calbiochem), chloroquine(CQ; Sigma-Aldrich), polybrene(Sigma-Aldrich), lipofectamine 2000(Invitrogen), puromycin(InvivoGen), EDTA-free protease inhibitor cocktail and phosphatase inhibitor cocktail(Roche Diagnostics), mouse monoclonal M2 FLAG affinity gel beads(Sigma-Aldrich), ammonium bicarbonate(NH₄HCO₃; Sigma-Aldrich), iodoacetamide(IAM; Sigma-Aldrich), formic acid(Sigma-Aldrich), Trifluoroacetic acid(TFA; Pierce), tris(2-carboxyethyl)phosphine(TCEP; Pierce), bovine trypsin(Roche Applied Science), acetonitrile(ACN;Thermo Fisher Scientific), POROS 20 R2 beads(Applied Biosystems), C18 ZipTips(Merck Millipore), isopropyl-β-D-thiogalactopyranoside(IPTG; Sigma-Aldrich), Dynabeads protein G(Invitrogen), protein G Sepharose(GE Healthcare Life Sciences), NuPAGE Bis-Tris and Tris-Acetate gels running system(Invitrogen), QuikChange Lightning Site-Directed Mutagenesis Kit(Agilent Technologies), Hybond-P PVDF membrane(GE Healthcare Life Sciences), BCA Protein Assay Reagent Kit(Pierce), Radioactive [γ-³²P]ATP(PerkinElmer), Calf intestinal alkaline phosphatase(New England Biolabs), Factor Xa(New England Biolabs), K48- and K63-linked poly ubiquitin chains(Boston Biochemical), DAPI-containing fluorescence mounting medium(Invitrogen) were purchased from indicated suppliers.

Common reagents were from Sigma-Aldrich (St. Louis, MO, USA). Alcohol or ethanol (200 proof, absolute ACS/USP Grade) was purchased from Pharmco Products Inc. (Shelbyville, KY, USA). Radioactive γ-³²P-ATP was obtained from Perkin Elmer (Billerica, MA, USA). All oligonucleotides were ordered from Integrated DNA Technologies (Coralville, IA, USA) and their sequences are displayed in Table 1.

Chemicals, growth media, and antibiotics were purchased from Thermo-Fisher (Whitby, Canada) or Sigma (Oakville, Canada), unless otherwise stated. Escherichia coli strains, such as NovaBlue and BL21(DE3), and other expression plasmids were purchased from EMD4 Biosciences (New Jersey, USA). All the primers were purchased from Sigma (Oakville, Canada). Chromatography columns and media were bought from GE Healthcare (Mississauga, ON, CA). Radioactive [γ-³²P]-ATP was purchased from Perkin Elmer (Life and Analytical Sciences, Waltham, MA, USA).