The NPCs were transfected with biotinylated miR-486-3p probe or biotinylated NC probe. After transfection for 48 h, the cells were incubated with cell lysate (Ambion, Austin, Texas, USA) for 10 min. The cleavage was incubated with beads precoated with M-280 streptavidin (Sigma) at 4 ℃ for 3 h. Then, the bound RNA was purified by Trizol (Boyetime), and the circ-STC2 or TFR2 expression was measured with RT-qPCR.
M 280 streptavidin
Manufactured by Merck Group
Sourced in United States
The M-280 streptavidin is a lab equipment product by Merck Group. It is a paramagnetic bead coated with streptavidin, a protein that binds strongly to biotin. The M-280 streptavidin is designed for use in various biological and biochemical applications that require the capture, separation, or detection of biotinylated molecules.
Automatically generated - may contain errors
4 protocols using m 280 streptavidin
1
Biotinylated miR-486-3p Pulldown Assay
2
Biotin-labeled miRNA Interactome Identification
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Three diverse biotin-labeled miRNA sequences WT miR-24-3p (Bio-miR-24-3p-WT), MUT miR-24-3p (Bio-miR-24-3p-MUT, sequence mutation that complementary to MCM3AP-AS1), and a random miRNA that did not complementary to MCM3AP-AS1 (Bio-NC) were designed and synthesized by GenePharma. The miRNAs were transfected into VECs for 48 h when the cell confluence reached 80–90%. Subsequently, cells were lysed using lysis buffer and the lysate was co-cultured with magnetic beads coated with M-280 streptavidin (Sigma-Aldrich Chemical Company, MO, USA) at 4°C for 3 h. The beads were rinsed and protein-nucleotide complex absorbed by the beads were eluted. Trizol was used to extract the total RNA, and MCM3AP-AS1 expression was assessed using RT-qPCR.
3
Quantifying Biotin-labeled miR-34a-3p Interactions
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Biotin-labeled miR-34a-3p WT and MUT plasmids (50 nM each) were transfected into cells severally. After 48-h transfection, the cells were incubated with a specific cell lysate (Ambion, Austin, Texas, USA) for 10 min. Then 50 mL of sample cell lysate was divided. Residual lysates were incubated with M-280 streptavidin magnetic beads precoated with RNase-free and yeast tRNA (Sigma, MO, USA) at 4℃ for 3 h. The magnetic beads were washed with cold lysate twice, low salt buffer three times, and high salt buffer once. Antagonistic miR-34a-3p probes were set as NC. Total RNA was extracted by Trizol, and DDX11-AS1 and TRAF5 expression were tested by RT-qPCR.
4
Purification and Analysis of LINC00917 and NLRP1
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According to a previous study [28 (link)], the NPCs were treated with biotinylated miR-149-5p or NC probes. After transfection for 48 h, the NPCs were treated with cell lysate (Ambion, Austin, TX, USA) for 10 min. The cleavage was incubated with beads precoated with M-280 streptavidin (Sigma) at 4°C for 3 h. Then, the bound RNA was purified using Trizol (Boyetime), and LINC00917 or NLRP1 levels were measured by RT-qPCR.
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