After linearization of the plasmids, T7 RNA polymerase (Roche, Basel, Switzerland, Cat#10881767001) and biotin RNA labelling mix (Roche, Cat#11685597910) were used to synthesize transcripts of the MEG3 v2 full-length [20 (link)]. Then, the transcripts were treated with DNase I and EDTA. Meanwhile, proteins were extracted and lysed from PSCs. In vitro biotinylated RNAs (2 µg) were incubated with streptavidin beads overnight, and then pulled down the proteins by complex. The beads were washed five times with wash buffer. Then, the protein complexes associated with beads were analysed by mass spectrometry and western blot. The mass spectrometry results of the sense strand and antisense strand were compared to find the protein that specifically binds to MEG3.
Protocol detail
Identifying MEG3-Binding Proteins
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Biotin
Buffer
Dnase i
Edta
Mass spectrometry
Plasmids
Protein
Pscs
Rnas
Streptavidin
T7 rna polymerase
Western blot
Corresponding Organization : Center of Hubei Cooperative Innovation for Emissions Trading System
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Variable analysis
independent variables
- Linearization of the plasmids
- Use of T7 RNA polymerase (Roche, Basel, Switzerland, Cat#10881767001) to synthesize transcripts of the MEG3 v2 full-length
- Use of biotin RNA labelling mix (Roche, Cat#11685597910) to synthesize transcripts of the MEG3 v2 full-length
- Incubation of in vitro biotinylated RNAs (2 µg) with streptavidin beads overnight
- Protein complexes associated with the beads that were analyzed by mass spectrometry and western blot
- Comparison of the mass spectrometry results of the sense strand and antisense strand to find the protein that specifically binds to MEG3
- Treatment of the transcripts with DNase I and EDTA
- Extraction and lysis of proteins from PSCs
- None specified
- None specified
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