Purified GST-HRS1 recombinant protein was used to determine DNA binding by EMSA. ssDNA oligonucleotides (listed in Supplementary Data 1 ) were biotin labeled using the Biotin 3’ End DNA Labeling Kit (Thermo Scientific) and complementary pairs were annealed to make dsDNA probes. The binding of recombinant protein (50 ng) to the biotin labeled probes (20 fmol) was carried out in a reaction mixture containing 10mM Tris, 50mM KCl, 1mM DTT, pH7.5, 2.5% glycerol, 5mM MgCl2, 1μg poly (dI-dC) and 0.05% NP-40. After incubation at 22°C for 30min the protein-probe mixture was separated in a 4% polyacrylamide native gel and transferred to a Biodyne B Nylon membrane by capillary action in 20xSSC buffer overnight (Thermo Scientific). After UV-crosslinking (254nm) for 90sec at 120mJ .cm−2, the migration of biotin-labeled probes was detected using streptavidin-horseradish peroxidase conjugates in the Chemioluminescent Nucleic Acid Detection Module (Thermo Scientific) and exposed to X-ray film. As a negative control, we tested that the GST tag has no affinity for HRS1 related CREs (Supplementary Fig. 5 ).
Chemioluminescent nucleic acid detection module
Manufactured by Thermo Fisher Scientific
The Chemioluminescent Nucleic Acid Detection Module is a laboratory equipment designed for the detection of nucleic acids through chemiluminescent techniques. It provides a platform for the analysis and quantification of DNA, RNA, or other nucleic acid targets.
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2 protocols using chemioluminescent nucleic acid detection module
1
EMSA Assay for HRS1 DNA Binding
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EMSA Assay for HRS1 DNA Binding
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Purified GST-HRS1 recombinant protein was used to determine DNA binding by EMSA. ssDNA oligonucleotides (listed in Supplementary Data 1 ) were biotin labeled using the Biotin 3’ End DNA Labeling Kit (Thermo Scientific) and complementary pairs were annealed to make dsDNA probes. The binding of recombinant protein (50 ng) to the biotin labeled probes (20 fmol) was carried out in a reaction mixture containing 10mM Tris, 50mM KCl, 1mM DTT, pH7.5, 2.5% glycerol, 5mM MgCl2, 1μg poly (dI-dC) and 0.05% NP-40. After incubation at 22°C for 30min the protein-probe mixture was separated in a 4% polyacrylamide native gel and transferred to a Biodyne B Nylon membrane by capillary action in 20xSSC buffer overnight (Thermo Scientific). After UV-crosslinking (254nm) for 90sec at 120mJ .cm−2, the migration of biotin-labeled probes was detected using streptavidin-horseradish peroxidase conjugates in the Chemioluminescent Nucleic Acid Detection Module (Thermo Scientific) and exposed to X-ray film. As a negative control, we tested that the GST tag has no affinity for HRS1 related CREs (Supplementary Fig. 5 ).
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