Magnetic rna protein pull down kit

RBPs pull-down assay was performed following the user guidelines of Magnetic RNA-Protein Pull‐Down Kit (Thermo Scientific Pierce, Waltham, USA), with some modifications. Briefly, S. suis cultures (20 mL) were collected and bacterial cells were resuspended with 1.5 mL phosphate buffer (50 mM, PH7.0), and sonicated for 15 min. After centrifugation at 8,000 × g for 10 min, the supernatants were collected and the concentration of total protein was quantified via BCA assay. About 100 μg protein per sample was used for pull-down assay. At the same time, 5 μg of bait RNA sample were denatured at 85°C for 5 min and then cooled immediately on ice. RNAs were labeled by desthiobiotinylation with the Pierce RNA 3′End Desthiobiotinylation Kit (Thermo Scientific Pierce, Waltham, USA), then incubated with Streptavidin Magnetic Beads. Then, these beads were mixed with total protein in RIP buffer at 4°C with agitation or rotation for 1 h to achieve RNA-binding proteins. After washing with RIP wash buffer three times, the final retrieved proteins were boiled in SDS loading buffer, separated by SDS-PAGE, subjected to silver staining, and the band of interest was excised and subjected to in-gel trypsinization. The tryptic peptides were analyzed by LC-MS/MS. Peptide mass fingerprint and sequence data were analyzed using the UniProt and NCBI databases.

RNA pull-down assay was carried out using Magnetic RNA-Protein Pull-Down Kit (Thermo Scientific Pierce). Label the target RNA using the included Thermo Scientific Pierce RNA 3′ Desthiobiotinylation Kit. 25–100 pmol of labeled RNA was bound to Streptavidin Magnetic Beads. Add 400 μL of Master Mix containing A549 nuclear lysate to the RNA-bound beads. Add 50 μL of Elution Buffer to the beads and mix well by vortexing. The eluted proteins were subjected to silver-staining (Invitrogen Silver Staining Kit, Thermo) and whole bands were excised and sent for LC-MS/MS analysis which was performed by Shanghai Luming Biological Technology Co., Ltd.

Cell lysates were prepared using RNA immunoprecipitation (RIP) solution and then equally allocated into several tubes (one tube used as input) and stored at -80°C for subsequent experiments. According to the instructions of the Magnetic RNA-protein Pull-down Kit (Pierce Biotechnology Inc, Rockford, IL, USA), 1 μg of biotin-labeled RNA was added with 500 μL of structure buffer, water bathed at 95°C for 2 min and then ice bathed for 3 min. When the full resuspension of the magnetic beads was observed, the EP tube was added with 50 μL of magnetic bead suspension, incubated at 4°C overnight and subsequently centrifuged at 1610 g for 3 min with the removal of the supernatant. Later, the suspension was washed with 500 μL RIP wash buffer for 3 times, added with 10 μL of cell lysis buffer and allowed to stand at room temperature for 1 h. Lastly, the incubated proteins in the magnetic RNA-protein mixture were eluted, and the protein concentration was detected by bicinchoninic acid (BCA) assay and protein expression determined by western blot analysis.

RNA pull-down was performed using a Magnetic RNA-Protein Pull-Down Kit (Pierce Biotechnology, USA) in accordance with the manufacturer's instructions.

RNA pull-down assay was performed with Magnetic RNA-Protein Pull-Down Kit (Pierce Biotechnology, Rockford, IL, USA) according to the protocol of manufacturer. The enriched proteins were recovered and detected by Western blotting.

RNA pull-down was conducted in MAD-MB-231 and MCF7 cells (1 × 10⁴) using the Magnetic RNA-Protein Pull-Down Kit (Pierce Biotechnology, Rockford, IL, USA) in accordance with the manufacturer's instructions. miR-641 probe and NC probe are constructed by sigma. The cells were harvested and lysed, and the supernatant was obtained by centrifugation (5000 rpm/min, 10 min). miR-641 probe and NC probe were incubated with a supernatant at 37°C overnight. The magnetic beads (Pierce Biotechnology) were then added to the supernatant and incubated for 1 h at 37°C to allow the beads to adsorb the probe. After cleaning, the mRNA binding to the probe was harvested. The relative NUCKS1 expression was analyzed by real-time PCR assay.