To obtain the biotin-labeled full-length or truncated Lionheart, we used CUGA® 7 in vitro Transcription kit (NIPPON GENE). When the biotin-labeled RNAs were synthesized, biotin-16-UTP (Sigma-Aldrich) was added, as the ratio of biotin-16-UTP to usual UTP was 3:2. The PCR product containing T7 promoter was employed as the template. The quantity and the quality of the synthesized RNAs were confirmed by NanoDrop™ 2000 spectrophotometer (Thermo Fisher Scientific) and Agilent 2100 bioanalyzer (Agilent Technologies). The RNAs were saved at −80 °C until use. To identify the Lionheart-specific-binding proteins, we used the biotin-labeled Lionheart antisense (AS) as the control.
Biotin 16 utp
Manufactured by Merck Group
Sourced in United States
Biotin-16-UTP is a modified form of the nucleotide uridine triphosphate (UTP), where a biotin molecule is attached to the ribose ring at the 16th carbon position. This modified nucleotide can be incorporated into RNA during in vitro transcription or labeling experiments.
Automatically generated - may contain errors
Lab products found in correlation
Dynabeads m 280 streptavidin, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Rnase inhibitor, by Thermo Fisher Scientific (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Cuga 7 in vitro transcription kit, by Nippon Gene (1 mentions)
Hybond p membrane, by GE Healthcare (1 mentions)
Mouse anti gapdh mab, by Merck Group (1 mentions)
Rneasy mini kit, by Thermo Fisher Scientific (1 mentions)
Pierce magnetic rna protein pull down kit, by Thermo Fisher Scientific (1 mentions)
Ez magna rip kit, by Merck Group (1 mentions)
Hiscribe t7 high yield rna synthesis kit, by New England Biolabs (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
T7 rna polymerase, by Thermo Fisher Scientific (1 mentions)
Phenol chloroform isoamyl alcohol 25 24 1, by Merck Group (1 mentions)
M13mp18 dna, by New England Biolabs (1 mentions)
Rna sample loading buffer, by Merck Group (1 mentions)
Chemiluminescent nucleic acid detection module, by Thermo Fisher Scientific (1 mentions)
6 protocols using biotin 16 utp
1
Synthesis and Characterization of Biotin-Labeled Lionheart RNA
2
Protein Isolation and Immunoblotting Protocol
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Protein isolation was performed using Cellytics MT Cell Lysis Reagent (Sigma-Aldrich). We separated equal amounts of protein on 5–20% SDS-PAGE gels (FUJIFILM Corp.) and transferred them to Hybond-P membranes (GE Healthcare). The following primary antibodies were used with indicated dilutions: rabbit anti-AR pAb (H-280, Santa Cruz Biotechnology., 1:2000), mouse anti-Gapdh mAb (Millipore, 1:5000), rabbit anti-MID1 pAb (Abcam, 1:1000), rabbit anti-PP2Ac pAb (Cell Signaling Technology, 1:1000), rabbit anti-GFP pAb (MBL, 1:1000). The density of each band was quantified using ImageJ software (NIH, Bethesda, MD, USA). For RNA pull-down assay, cell lysate containing ~300 μg of total protein derived from HEK293T cells overexpressed with Mid1 was incubated for 1 h with 3 μg of in vitro-transcribed RNA biotinylated with Biotin-16-UTP (Sigma-Aldrich). It was followed by incubation with Dynabeads M-280 streptavidin (Invitrogen) for 1 h at 4 °C and washed with wash buffer three times.
3
Nuclear Run-on Assay with RNA Pulldown
4
RNA-Protein Interactome Profiling
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RNA pulldown assay was carried out under the guidance of the instruction provided by the Pierce Magnetic RNA-Protein Pull-Down Kit (#20164, Thermo Scientific). Briefly, Biotin-16-UTP (#11388908910, Sigma-Aldrich, USA) and HiScribe T7 High Yield RNA Synthesis Kit (#E2040S, NEB, USA) were adopted to transcribe specific plasmids into biotin-labeled RNAs, including sense and antisense sequences. Which were then bound to the streptavidin magnetic beads, and incubated overnight with the nuclear fraction obtained by the subcellular protein fractionation kit. The RNA-binding proteins were eluted from the beads and analyzed by western blotting or silver staining.
RNA immunoprecipitation assay was performed following the instruction of the EZ-Magna RIP kit (#17-700, Sigma-Aldrich). Cell lysates were obtained using the RIP lysis buffer, and incubated with the RIP buffer containing antibody-conjugated magnetic beads overnight. The immunoprecipitated RNAs were extracted with TRIzol reagent and analyzed by qPCR.
RNA immunoprecipitation assay was performed following the instruction of the EZ-Magna RIP kit (#17-700, Sigma-Aldrich). Cell lysates were obtained using the RIP lysis buffer, and incubated with the RIP buffer containing antibody-conjugated magnetic beads overnight. The immunoprecipitated RNAs were extracted with TRIzol reagent and analyzed by qPCR.
5
Transcriptional Regulation of PLEKHA7 Expression
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A nuclear run-on assay was performed using previously described methods [46 (link)] to determine whether PLEKHA7 expression induced by p50 or hTERT was caused by transcriptional regulation. Nuclei were extracted from MKN74 cells (5◊107) after different treatments in NP-40 lysis buffer. The nuclear extracts were resuspended in 500 µL of nuclear freezing buffer and then mixed thoroughly with 200 µL of 5◊run-on buffer. Then, 8 µL of biotin-16-UTP (11388908910, Sigma–Aldrich) were added to the nuclei, and the sample was incubated for 20 min at 37 °C. The nuclear run-on reaction was quenched with 1.5 µL of 1 M CaCl2. RNA was purified with an RNeasy mini kit (74104, QIAGEN) and divided into small aliquots. The RNA samples were mixed with Dynabeads M-280 with streptavidin (11205D, Thermo Fisher Scientific) and incubated at 37 °C for 2 h on a rotator. Then, the RNA samples were washed with 2× saline sodium citrate (SSC) 3 times, and the beads were isolated and resuspended in 20 µL of nuclease-free water. RT–qPCR was performed, and the PCR products were separated on a 1.5% agarose gel.
6
In Vitro Transcription Assay on ssDNA
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The in vitro transcription assay on ssDNA template was carried out as described previously (Wanrooij et al., 2008) . The 20 l reaction mix containing 80 ng of single-stranded M13mp18 DNA (New England Biolabs), 10 mM Tris HCl (pH 8.0), 100 g/ml BSA, 20 mM MgCl2, 1 mM DTT, 1 mM each ATP, CTP and GTP, 0.65 mM UTP, 0.35 mM Biotin-16-UTP (Sigma), 10 units of RNase inhibitor (Thermo Fisher Scientific), with the indicated amount of T7 RNA polymerase (Ambion) or 500 fmol of purified recombinant proteins was incubated at 32 °C for 30 min. The RNA products were extracted twice with Phenol:
Chloroform: Isoamyl Alcohol 25:24:1 (Sigma) and precipitated with 2.5 -3.0 volumes of ethanol for 1 h to overnight at -20 °C. The samples were dissolved in RNA sample loading buffer (Sigma), denatured for 5 min at 75 °C, and analyzed on a denaturing polyacrylamide gel. The products were detected by the Chemiluminescent Nucleic Acid Detection Module (Thermo Fisher Scientific). To semi-quantify the level of in vitro transcription, the total densitometry of Biotin-16-UTP chemiluminescence in each lane was measured and normali ed to that of 0.1 pmol 5 -Biotin-ssRNA, which was loaded on the same gel and hence used as the internal standard. The relative level of Biotin-16-UTP in each reaction was calculated and plotted.
Chloroform: Isoamyl Alcohol 25:24:1 (Sigma) and precipitated with 2.5 -3.0 volumes of ethanol for 1 h to overnight at -20 °C. The samples were dissolved in RNA sample loading buffer (Sigma), denatured for 5 min at 75 °C, and analyzed on a denaturing polyacrylamide gel. The products were detected by the Chemiluminescent Nucleic Acid Detection Module (Thermo Fisher Scientific). To semi-quantify the level of in vitro transcription, the total densitometry of Biotin-16-UTP chemiluminescence in each lane was measured and normali ed to that of 0.1 pmol 5 -Biotin-ssRNA, which was loaded on the same gel and hence used as the internal standard. The relative level of Biotin-16-UTP in each reaction was calculated and plotted.
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