Recombinant GST-BNIP3 and GST-BNIP3L was produced in BL21 competent cells grown overnight and treated with IPTG for 4 h. Recombinant protein was purified using a Glutathione Sepharose 4B bead slurry and eluted via thrombin cleavage. In vitro kinase assays were performed using recombinant ULK1 (ThermoFisher PV6430), 5X Kinase Buffer A (ThermoFisher PV3189), recombinant BNIP3/BNIP3L, ATP (0.2 mM), ULK-101 (0.5 μM), and ATP [γ-32P] (PerkinElmer BLU002Z250UC). Assays were incubated at 37 °C for 30 min, diluted 1:1 in 2 × sample loading buffer, and boiled for 5 min. Samples were loaded onto an SDS-PAGE gel overnight, followed by gel drying and visualization by exposure on X-ray film.
Blu002z250uc
Manufactured by PerkinElmer
The BLU002Z250UC is a lab equipment product from PerkinElmer. It is a compact and versatile device that can be used for various laboratory applications. The core function of this product is to provide a reliable and consistent performance for the user's needs. For a detailed and unbiased description, please consult the product's technical specifications or speak with a PerkinElmer sales representative.
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5 protocols using blu002z250uc
1
Recombinant BNIP3/BNIP3L Kinase Assay
2
DNA Probe Labeling and Purification
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The 24 nt DNA probe (Supp. Table 1 ) hybridizes to pseudoknot nt 130–153. 10 pmol probe oligo was 5′ 32P labeled using 10 μL γ32P-ATP (PerkinElmer BLU002Z250UC). The reaction was carried out in 1 x PNK Buffer (NEB) at a total volume of 20 μL with 10 U of PNK (NEB). The probe was purified using two sequential Centri-Spin 10 columns following the manufacturer’s protocol.
3
Probing p53 Binding to PD-1 Promoter
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The probe that containing p53-binding element of PD-1 promoter was labeled with 32P (PerkinElmer, BLU002Z250UC) through a T4 polynucleotide kinase–mediated 5′ end phosphorylation reaction (NEB, M0201), and the labeled probe was then purified using Bio-Spin column (Bio-Rad, 732-6223) according to the manufacturer’s protocol. For shift assay, the labeled probe together with or without the cold probe (unlabeled wild-type or mutated probe) was incubated with purified p53 in 1× EMSA buffer [10 mM Hepes (pH 7.6), 40 mM NaCl, 50 μM EDTA, 6.25% glycerol, 1 mM MgCl2, 1 mM spermidine, 1 mM dithiothreitol (DTT), bovine serum albumin (BSA; 50 ng/μl), and sheared single-strand salmon DNA (5 ng/μl)] for 20 min at room temperature. For supershift assay, α-p53 antibody (Santa Cruz Biotechnology, sc-126) was preincubated with purified p53 in the reaction system without probe for 30 min at room temperature, and then the labeled probe was added for further 20-min incubation. The complex was separated by 4% tris-borate EDTA–PAGE and visualized by autoradiography.
4
DNA Probe Labeling and Purification
5
Quantifying ATP Hydrolysis Kinetics
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Radioactive γ-P32-ATP (PerkinElmer, BLU002Z250UC) was added to 600 μM MgATP (pH = 7.0) solutions at volume ratios of 1:1000-1:300, depending on the lifetime of the radioactive reagent. The total volume of each reaction was 12 μL, including 6 μl of protein from size exclusion chromatography fractions (final concentration ∼0-50 nM for different fractions, peak fractions were used for Rbin-1 and AMPPNP inhibition experiments in Figure 3 E), 4 μl FPLC SEC buffer with 0.6 mM Na2SO4 and 2 μl MgATP (final concentration = 100 μM). The reactions were then incubated at room temperature for 30 or 60 min before quenching with 12 μl 0.2 M EDTA. 1 μl from each reaction mixture was spotted on to TLC PEI cellulose F plates (Millipore, 105579). The TLC buffer contained 0.15 M formic acid and 0.15 M lithium chloride. The TLC plates were then imaged using the Typhoon Scanner 9400 (GE Healthcare Life Sciences). ImageJ was used to calculate the densitometric ratio of the spots corresponding to radioactive free phosphate and ATP to determine the percent of ATP hydrolyzed.
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