In vitro kinase assays were performed using GST-tagged FANCA fragments 1–4 (GST-FANCA-F1 to -F4), as described previously [17 (link)]. The purified GST-tagged FANCA fragments were incubated with recombinant AMPKα (72 ng, Cell Signaling #7464) in kinase buffer comprised of 60 mM HEPES (pH 7.4), 3 mM MgCl2, 3 mM MnCl2, 1.2 mM DTT, 50 μM cold ATP, and 2 μCi [γ-P32] ATP (Perkin Elmer, Waltham, MA, USA) at 30°C for 30 min. Samples were subjected to SDS-PAGE, transferred to nitrocellulose membranes (GE Healthcare, Milwaukee, WI, USA), and visualized by autoradiography.
γ p32 atp
Manufactured by PerkinElmer
Sourced in United States, Germany
[γ-P32] ATP is a radioactive compound that contains the isotope phosphorus-32 (P-32) in the gamma phosphate position of adenosine triphosphate (ATP). It is used as a tracer in various biochemical and molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
T4 polynucleotide kinase, by New England Biolabs (3 mentions)
Nitrocellulose membrane, by GE Healthcare (2 mentions)
Bas ip ms 2340, by Fujifilm (2 mentions)
Imagequant 5, by GE Healthcare (2 mentions)
Phosphorimaging screen, by GE Healthcare (2 mentions)
Tlc pei cellulose f paper, by Merck Group (2 mentions)
Unlabeled atp, by Merck Group (2 mentions)
Polynucleotide kinase, by New England Biolabs (1 mentions)
Adenosine triphosphate (atp), by PerkinElmer (1 mentions)
12 protocols using γ p32 atp
1
FANCA Phosphorylation by AMPK In Vitro
2
RNase H Hydrolysis Assay of Modified RNA
3
Radiolabelled Probe Preparation for EMSA
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Radiolabelled probe for EMSA were prepared by end labelling with [γ-P32] ATP (3000 Ci/mmol, Perkin Elmer), using T4 polynucleotide kinase (New England Biolabs), and separated from free nucleotide by a sephadex G-50 spin column. [γ-P32] ATP labelled probe was incubated with purified FvCamlp in 5X binding buffer (20% glycerol, 5 mM MgCl2, 2.5 mM EDTA, 2.5 mM DTT, 250 mM NaCl, 50 mM Tris-HCl (pH 7.5), 0. 25 mg/ml of poly dA-dT) at room temperature for 30 mins. Protein-DNA complexes and free DNA were fractionated on 5% polyacrylamide gels in 1xTBE buffer pH 8.3, at 4 °C and visualized by autoradiography.
4
Preparation of DNA Substrates for Biochemical Assays
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The Φx174 viral (+) strand ssDNA and replicative form I dsDNA were purchased from New England BioLabs. Oligonucleotides were gel-purified as previously described (29 (link)). To prepare 5′-end-labeled ssDNA for Benzonase protection assays, 80-mer Oligo 1 was incubated with polynucleotide kinase (New England Biolabs) and [γ-P32-ATP] (PerkinElmer) for 5′-end-labeling. The unincorporated nucleotide was removed using a Spin 6 column (Bio-Rad). For oligonucleotide-based DNA binding, the DNA substrates Oligos 2–4 and Oligos 5–10 were derived from previous researches (30 (link),31 (link)). The sequences of DNA substrates used in our DNA mobility shift and Benzonase protection assays are summarized in Supplementary Table S1 , and the composition of annealed DNA substrates for oligonucleotide-based DNA binding is summarized in Supplementary Table S2 . For tethering particle motion analysis, a gapped DNA substrate (151 bp dsDNA handle + (dT)135 nt ssDNA + 19 bp dsDNA handle) was prepared by annealing the PCR products as described in previous reports (32 (link),33 (link)).
5
MACC1-MEK1 Kinase Assay Protocol
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Ten micrograms MACC1-GFP from Nanotrap IP were incubated with 500-ng recombinant MEK1 S218D S222D (Thermo Fisher) and 0.3-µM γP32 ATP (Perkin Elmer, Rodgau, Germany) at 37 °C in 80-mM HEPES pH 7.5, 4-mM MgCl2, 4-mM MnCl2, 1.6-mM DTT, and 70-µg/ml PEG20 000 for 15, 30, 45, and 60 min. The reaction was competed with 1000 × cold ATP or inhibited with 1.4-µM AZD6244. Heat-inactivated samples were separated with 10% polyacrylamide gels. The gels were dried and developed with imaging plates (BAS-IP MS 2340, Fujifilm, Japan).
6
In Vitro Phosphorylation of GIGYF1
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GFP-tagged GIGYF1 was immunopurified from transfected U2OS cells lysed in high-salt EBC buffer (50 mM Tris, pH 7.5; 500 mM NaCl; 1 mM EDTA; 0.5% NP40; 1 mM dithiothreitol (DTT)). Beads were washed extensively with lysis buffer and once with kinase buffer (25 mM HEPES, pH 7.2; 25 mM MgCl; 2 mM DTT). Reactions were initiated by adding 450 ng recombinant MK2 (Abcam) and 25 mM ATP spiked with γ-P32-ATP (10 mCi, Perkin Elmer) to each sample. Indicated samples were supplemented with 100 ng recombinant HSP27 (Prospec, Rehovot, Israel) to serve as positive control for MK2 activity. Reactions were incubated for 30 min at 30 °C with gentle shaking and terminated by adding Laemmli buffer and boiling the samples at 95 °C for 10 min. Samples were then run on SDS-PAGE and vacuum-dried on a Whatman filter paper (Sigma). Relative phosphorylation was assayed by autoradiography.
7
Radiolabeled DNA Translocation Assay
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CM0082-IB (5′-CTGGCTGTGGCGTGTTTCTGGTGG[bio-dT]ACCTAGGTCTTAGCCGTCTACGCCTCACT-3′) was radiolabeled with T4 Polynucleotide Kinase (New England Biolabs) in the presence of [γP32]ATP (PerkinElmer). For dsDNA assays, radiolabeled CM0082-IB was incubated in fivefold molar excess CM0082-RC-IB (5′-AGTGAGGCGTAGACGGCTAAGACCTAGG[bio-dT]ACCACCAGAAACACGCCACAGCCAG-3′, heated to 95 °C for 2 min, and slowly cooled to room temperature to anneal the two strands. Single-stranded radiolabeled CM0082-IB or radiolabeled double-stranded CM0082-IB/CM0082-RC-IB was bound to 40-fold molar excess streptavidin. Translocase reactions were assembled with final concentrations of 5 nM ssDNA or dsDNA substrate and 15 nM Eta in 20 mM Tris⋅HCl, pH 8.0, 10 mM MgCl2, 10 μg/mL bovine serum albumin (BSA), and 1 mM dithiothreitol (DTT). Reactions were heated to 50 °C and started by addition of dATP and excess biotin (400 µM) to trap streptavidin displacement events. At the appropriate time points, 10-μL aliquots were removed and stopped on ice with the addition of 3 μL 1.5% SDS and 50% glycerol. Terminated reactions were separated on 15% native polyacrylamide gels, and radiolabeled products were detected by exposure of the gel to a phosphorimaging screen (GE Healthcare) and analyzed using GE Imagequant 5.2.
8
Helicase-Catalyzed Unwinding of 3'-overhung dsDNA
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The short DNA strand of the 3′-overhung dsDNA substrate (CM0080; 5′-GACCTAGGAACCACCAGAAACACGCCACAGCCAG-3′) was radiolabeled with T4 Polynucleotide Kinase (New England Biolabs) in the presence of [γP32]ATP (PerkinElmer). Equal molar amounts of CM0080 and the long DNA strand of the 3′-overhung dsDNA substrate (CM0082; 5′-CTGGCTGTGGCGTGTTTCTGGTGGTTCCTAGGTCTTAGCCGTCTACGCCTCACT-3′) were mixed, heated to 95 °C for 2 min, and slowly cooled to room temperature to anneal the two strands, forming the substrate. Helicase reactions were assembled with final concentrations of 5 nM substrate and 15 nM Eta in 20 mM Tris⋅HCl, pH 8.0, 10 mM MgCl2, 10 μg/mL BSA, and 1 mM DTT. Reactions were heated to 50 °C and started by addition of dATP and trap DNA (CM0079; 5′-CTGGCTGTGGCGTGTTTCTGGTGGTTCCTAGGTC-3′) to 3.5 mM and 25 nM, respectively. At the appropriate time points, 10-μL aliquots were removed and stopped on ice with the addition of 3 μL 1.5% SDS and 50% glycerol. Terminated reactions were separated on 15% native polyacrylamide gels, and radiolabeled products were detected by exposure of the gel to a phosphorimaging screen (GE Healthcare) and analyzed using GE Imagequant 5.2.
9
In Vitro Kinase Assay of FANCA Fragments
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The in vitro kinase assay was performed using four previously described GST‐tagged FANCA fragments (#1 to #4) 11 . Purified GST‐tagged FANCA fragments were incubated with 85 ng of Aurora A kinase protein (Cell Signaling Biotechnology, Danvers, MA, USA) in kinase buffer composed of 25 mm Tris–HCl (pH7.4), 10 mm MgCl2, 2 mm DTT, 200 μm cold ATP, and 5 μCi [γ‐P32] ATP (Perkin Elmer, Waltham, MA, USA) at 30 °C for 30 min. Samples were subjected to SDS/PAGE and transferred to nitrocellulose membranes (GE Healthcare, Milwaukee, WI, USA), followed by autoradiography to detect phosphorylated protein.
10
Kinase Assay for DCLK1-Mediated MACC1 Phosphorylation
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For the kinase assay, DCLK1 was isolated from HEK293T cells after lentiviral transduction. To isolate the protein, Nanotrap-based (ChromoTek, Martinsried, Germany) Co-IP for GFP was performed following manufacturers recommendations. Cleared lysates of 107 cells were incubated with Nanotrap-coupled beads for 1 h. After washing, the protein-loaded beads were directly used for the kinase assay. Five-hundred nanograms of recombinant MACC1 (Origene, Rockville, MD, USA) was incubated at 37 °C with 10 µg isolated DCLK1 in kinase buffer containing 0.3 µM γP32 ATP (Perkin Elmer, Rodgau, Germany), 80 mM HEPES pH 7.5, 4 mM MgCl2, 4 mM MnCl2, 1.6 mM DTT, and 70 µg/mL PEG20,000 for 15, 30, 45, and 60 min. Samples were heat-inactivated and separated on 10% polyacrylamide gels. The gels were dried on Whatman paper and bands were visualized with imaging plates (BAS-IP MS 2340, Fujifilm, Japan) using an Amersham Typhoon FLA 7000 (GE Healthcare, Freiburg, Germany).
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