Nuclease assays were performed as previously (Anand et al., 2016 (link)). Briefly, proteins were added as indicated, in volume of 15 μL in reaction buffer containing Tris-acetate [pH 7.5], 25 mM; manganese acetate, 1 mM; magnesium acetate, 5 mM; dithiothreitol, 1 mM; ATP, 1 mM; rNTPs, 83 μM; BSA, (New England Biolabs), 0.25 mg/mL; phosphoenolpyruvate, 1 mM; pyruvate kinase, (Sigma), 80 U/mL; DNA substrate (Table S1 ), 1 nM and streptavidin (Sigma), 15 nM. Recombinant proteins were then added to the reactions on ice and the samples were incubated for 30 min at 37°C. Reactions were stopped and separated on 15% polyacrylamide denaturing urea gels (19:1 acrylamide: bisacrylamide, Bio-Rad). The gels were fixed for 30 min at room temperature, dried and exposed to storage phosphor screens (GE Healthcare) and scanned by a Typhoon Phosphor imager (FLA 9500, GE Healthcare).
Typhoon phosphor imager fla 9500
Manufactured by GE Healthcare
The Typhoon Phosphor Imager FLA 9500 is a high-performance fluorescence and phosphor imaging system designed for diverse applications in life science research. It provides sensitive and quantitative detection of various fluorescent and phosphor-labeled samples.
Automatically generated - may contain errors
Lab products found in correlation
Storage phosphor screen, by GE Healthcare (6 mentions)
17 chr chromatography paper, by Cytiva (2 mentions)
Proteinase k, by Roche (2 mentions)
Bovine serum albumin (bsa), by New England Biolabs (2 mentions)
Phosphoenolpyruvate, by Merck Group (1 mentions)
Pyruvate kinase, by Merck Group (1 mentions)
T4 polynucleotide kinase, by PerkinElmer (1 mentions)
γ 32p atp, by PerkinElmer (1 mentions)
3mm paper, by Cytiva (1 mentions)
De81 chromatography paper, by Cytiva (1 mentions)
7 protocols using typhoon phosphor imager fla 9500
1
Nuclease Assay Protocol for Protein Analysis
2
Oligonucleotide Radiolabeling and Purification
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The 5′-end-radiolabeled single-stranded oligonucleotides
were
prepared by reacting the DNA with T4 polynucleotide kinase (NEB) and
[γ-32P]ATP (PerkinElmer) for 45 min at 37 °C.
The radiolabeled DNA oligonucleotides were purified by 17% denaturing
PAGE and extracted from the gel via the crush and soak method. The
labeled aptamer was conjugated onto the nanoparticle. The samples
were analyzed on an 1% agarose gel. The gel was exposed to a phosphorimager
screen and then visualized by Typhoon Phosphorimager FLA 9500 (GE
Healthcare, Life Sciences).
were
prepared by reacting the DNA with T4 polynucleotide kinase (NEB) and
[γ-32P]ATP (PerkinElmer) for 45 min at 37 °C.
The radiolabeled DNA oligonucleotides were purified by 17% denaturing
PAGE and extracted from the gel via the crush and soak method. The
labeled aptamer was conjugated onto the nanoparticle. The samples
were analyzed on an 1% agarose gel. The gel was exposed to a phosphorimager
screen and then visualized by Typhoon Phosphorimager FLA 9500 (GE
Healthcare, Life Sciences).
3
In vitro Phosphorylation of BLM by CDK1, CDK2, and PLK1
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In vitro phosphorylation reactions were performed in 20-μl volume in kinase buffer [50 mM tris-HCl (pH 7.5), 5 mM magnesium acetate, 0.2 mM EDTA, 100 mM NaCl, 0.5 mM sodium orthovanadate, and 10 mM p-nitrophenyl phosphate]. A total of 0.3 μg of BLM, 40 ng of either CDK1-CycB or CDK2-CycA, 40 ng of PLK1, and 0.6 μg of TOPBP1 were added to the reaction, as indicated. All the components were mixed on ice. Reactions were initiated by the addition of 0.1 mM adenosine triphosphate (ATP) and 37 kilobecquerel of 5′-[γ-32P]-ATP (PerkinElmer) and incubated at 37°C for 30 min. Phosphorylated samples were then used for biochemical assays as described below or prepared for autoradiography. In the latter case, phosphorylation reactions were stopped by adding 5 μl of SDS buffer [50 mM tris-HCl (pH 6.8), 1.6% (m/w) SDS, 10% (v/v) glycerol, 0.1 M dithiothreitol (DTT), and 0.01% (w/v) bromophenol blue] and incubated at 95°C for 5 min. Samples were resolved by SDS–polyacrylamide gel electrophoresis, stained with Coomassie dye, and destained in 40% (v/v) methanol and 10% (v/v) acetic acid. The gel was dried on 3MM paper (Whatman), exposed to storage phosphor screen (GE Healthcare), and scanned using Typhoon Phosphor Imager FLA 9500 (GE Healthcare). BLM phosphorylation was performed with CDK1-CycB or CDK2-CycA, which modified BLM with similar efficiency.
4
DNA Unwinding Assay Protocol
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DNA unwinding assays (volume, 15 μl) were carried out in a reaction buffer containing 25 mM tris-acetate (pH 7.5), 5 mM magnesium acetate, 1 mM DTT, BSA (0.1 mg/ml), 1 mM phosphoenolpyruvate, pyruvate kinase (80 U/ml), 1 mM ATP, 100 mM NaCl, and 0.1 nM 32P-labeled oligonucleotide-based DNA substrate (in molecules). The reactions were assembled on ice, initiated by the addition of ATP, and incubated at 37°C for 30 min. Next, 5 μl of 2% STOP solution [150 mM EDTA, 2% (m/w) SDS, 30% (v/v) glycerol, and 0.1% (w/v) bromophenol blue] and 1 μl of proteinase K (20.3 mg/ml; Roche) was added to stop the reaction for 10 min at 37°C. The 2% STOP solution was supplemented with 20-fold molar excess of the X12-3 cold oligonucleotide to avoid the reannealing of the unwound products. The products were separated on 10% native polyacrylamide gel. The gels were transferred on 17 Chr chromatography paper (Whatman), dried, exposed to storage phosphor screens (GE Healthcare), and scanned using the Typhoon Phosphor Imager FLA 9500 (GE Healthcare).
5
DNA Resection Assay with PCR Substrate
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Resection assays with PCR-based DNA substrate were performed in 15-μl volume in 25 mM tris-acetate (pH 7.5), 2 mM magnesium acetate, 1 mM ATP, 1 mM DTT, BSA (0.1 mg/ml), 1 mM phosphoenolpyruvate, pyruvate kinase (80 U/ml), 100 mM NaCl, and 1 nM substrate (in molecules). Human RPA was included as indicated to saturate all ssDNAs. After the addition of the other recombinant proteins on ice, the reactions were incubated for 30 min at 37°C. Reactions were stopped by adding 5 μl of 2% STOP solution [150 mM EDTA, 2% (m/w) SDS, 30% (v/v) glycerol, and 0.1% (m/w) bromophenol blue] and 1 μl of proteinase K (20.3 mg/ml; Roche) and incubated at 37°C for 10 min. Samples were analyzed by 1% agarose gel electrophoresis. Gels were dried on DE81 chromatography paper (Whatman), exposed to storage phosphor screens (GE Healthcare), and scanned using the Typhoon Phosphor Imager FLA 9500 (GE Healthcare).
6
DNA-Binding Reactions Optimization Protocols
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The DNA‐binding reactions with plasmid DNA (15 μl) were carried out in a binding buffer containing 25 mM Tris‐acetate pH 7.5, 1 mM DTT, 0.1 mg/ml BSA, 3 mM EDTA, and 25 mM NaCl, with 100 ng of the plasmid DNA substrate. The indicated concentrations of recombinant proteins were added as the last components. The reactions were assembled and incubated on ice for 15 min, followed by the addition of 5 μl EMSA loading dye (50% glycerol, 0.01% bromophenol blue). The products were separated on 0.8% native agarose gels. Post electrophoresis, gels were stained in water containing GelRed (1:20,000) with gentle agitation for 45 min and imaged at InGenius3 (GeneSys). The oligonucleotide‐based DNA‐binding reactions (15 μl) were carried out in the binding buffer described above, with 0.5 nM (in molecules) radioactively labeled substrate. The reactions were supplemented with a competitor dsDNA (1.5 ng/μl of pUC19). The reactions were assembled in ice, incubated at 37°C for 30 min, followed by the addition of 5 μl EMSA loading dye. The products were separated on a native 6% polyacrylamide gel. Gels were dried on 17CHR chromatography paper (Whatman), exposed to storage phosphor screens (GE Healthcare), and scanned using the Typhoon Phosphor Imager FLA 9500 (GE Healthcare).
7
Dna2-E675A Binding Assay on Y-structure DNA
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DNA-binding reactions (15 µl volume) were carried out in 25 mM Tris-acetate pH 7.5, 5 mM magnesium acetate, 200 mM KCl, 1 mM dithiothreitol (DTT), 100 µg/ml bovine serum albumin (BSA, New England Biolabs), ssDNA substrate (oligonucleotide X12-3HJ328 , as indicated, 0.1 nM, in molecules). Proteins were added and incubated at 30 °C for 30 min. Loading dye (5 µl; 50% glycerol, bromophenol blue) was added to the reactions and the products were separated on 6% polyacrylamide gels (ratio acrylamide:bisacrylamide 19:1, Bio-Rad) in TAE buffer (40 mM Tris, 20 mM acetic acid and 1 mM EDTA) at 4 °C. The gels were dried on 17 CHR (Whatman), exposed to a storage phosphor screen (GE Healthcare) and scanned by a Typhoon Phosphor Imager (FLA 9500, GE Healthcare). Binding of Dna2-E675A to the Y structure (oligonucleotides X12-3HJ3 and X12-3TOPL, 0.1 nM, in molecules) was performed under identical conditions with 2 mM magnesium acetate and 1 mM ATP but without added salt.
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