Biotin labeled and unlabeled RNA oligonucleotides probes corresponding to the human cMYC ARE (1-6) and two random probes (R1, R2) were synthesized as indicated in Table S6 by Sigma-Aldrich. REMSA was performed with a LightShift chemiluminescent RNA EMSA Kit (Thermo Fisher Scientific) following the manufacturer’s instruction. Briefly, purified human FXR1-GST protein (5 mg/ml) (Novus Biologicals, USA) and purified human GST protein (5 mg/ml) (Sigma-Aldrich) were incubated for 30min with biotinylated probes (100 pM) in REMSA binding buffer and glycerol. In competition binding assays, unlabeled RNA oligonucleotides (Sigma-Aldrich) were added in increasing amounts (200-fold) and incubated for another 10 min at room temperature. RNA/protein complexes were then subjected to electrophoresis by 6% native polyacrylamide gel and transferred to nylon membrane (Thermo Fisher Scientific). RNA was cross-linked with a UV lamp at a distance of 0.5 cm from the membrane for 2 min. The membrane was blocked in blocking buffer for 15 min and replaced the blocking buffer with conjugate/blocking buffer. After washed with 1 × wash buffer for 3 times, membrane was incubated in substrate equilibration buffer for 5 min. Then, the membrane was incubated in working solution and exposed.
Unlabeled rna oligonucleotides
Manufactured by Merck Group
Unlabeled RNA oligonucleotides are single-stranded RNA molecules of varying lengths, typically ranging from a few to several dozen nucleotides. They are designed without any attached labels or modifications, allowing for maximum flexibility in downstream applications.
Automatically generated - may contain errors
2 protocols using unlabeled rna oligonucleotides
1
RNA-Protein Binding Assay with REMSA
2
RNA-Protein Binding Assay with REMSA
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Biotin labeled and unlabeled RNA oligonucleotides probes corresponding to the human cMYC ARE (1-6) and two random probes (R1, R2) were synthesized as indicated in Table S6 by Sigma-Aldrich. REMSA was performed with a LightShift chemiluminescent RNA EMSA Kit (Thermo Fisher Scientific) following the manufacturer’s instruction. Briefly, purified human FXR1-GST protein (5 mg/ml) (Novus Biologicals, USA) and purified human GST protein (5 mg/ml) (Sigma-Aldrich) were incubated for 30min with biotinylated probes (100 pM) in REMSA binding buffer and glycerol. In competition binding assays, unlabeled RNA oligonucleotides (Sigma-Aldrich) were added in increasing amounts (200-fold) and incubated for another 10 min at room temperature. RNA/protein complexes were then subjected to electrophoresis by 6% native polyacrylamide gel and transferred to nylon membrane (Thermo Fisher Scientific). RNA was cross-linked with a UV lamp at a distance of 0.5 cm from the membrane for 2 min. The membrane was blocked in blocking buffer for 15 min and replaced the blocking buffer with conjugate/blocking buffer. After washed with 1 × wash buffer for 3 times, membrane was incubated in substrate equilibration buffer for 5 min. Then, the membrane was incubated in working solution and exposed.
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