Classical flow of magnetic pull-down RNA pull-down
RNA pull-down using streptavidin magnetic beads (PuriMag G-strep product number PMG015)
RNA pull-down assay using Streptavidin magnetic beads
1. RNA pull-down Day 1 - Biotin probe-labeled RNA
1) Preparation: Remove the PierceTM RNA 3' End Desthiobiotinylation Kit, place the PEG and DMSO in the kit at room temperature, and melt the remaining components on ice. The PEG can also be thawed at 37 ° C; the PCR instrument is turned on;
2) According to the content of RNA master tube, RNA is dissolved in DEPC water, we usually 10ug/tube, and dissolved in 10μl DEPC water. Simultaneously dissolve the Control RNA in the kit as a negative control;
3) Take 5 μl of control RNA and target RNA, place in 200 μl PCR tube, add 1 μl DMSO to each tube, mix and place on a PCR machine, 85 ° C, 5 min, then immediately place on ice for 5 min. (The addition of DMSO can help open the secondary structure of RNA and improve the efficiency of RNA ligation)
4) Prepare a system for probe labeling reactions according to the following table:
Ingredient volume (μl)
Water 3
10X RNA ligase recation buffer 3
RNase inhibitor 1
Non-labeled RNA 5
Biotinylated Cytidine Bisphosphate 1
T4 RNA Ligase 2
PEG 30% 15
Total 30
5) Mix the above reaction system, then put it into the PCR machine at 16 ° C for 4 h to overnight. The extension of the reaction time can improve the connection efficiency;
6) After the completion of the reaction, add 400 μl of nuclease free-water to each tube;
7) Add 300 μl of phenol chloroform to each tube to extract successfully labeled RNA; centrifuge at maximum speed for 15 min after vortexing, carefully transfer the upper aqueous phase to a new EP tube;
8) Add 10 μl of 5 M NaCl, 2 μl of glycogen, and 600 μl of pre-cooled 100% ethanol, and precipitate at -20 ° C or -80 ° C to precipitate overnight.
2. RNA pull-down the next day
1) Preparation
· DTT, protein triplet dissolved at room temperature;
Streptavidin magnetic beads; (S1420, New England Biolabs)
·RNase Inhibitor;
· Cells, 10cm large dish, up to about 80~90% density;
2) RNA extraction
a) The precipitated overnight RNA was taken out, centrifuged at 4 ° C, the maximum speed for 30 min, and the white precipitate at the bottom of the tube was observed after centrifugation, and was RNA;
b) Carefully remove the supernatant, wash it with 1ml of 70% ethanol, centrifuge at 8000rpm for 10min, then absorb the liquid in the tube and dry the RNA in the air. If the ethanol is not completely evaporated, it will affect the efficiency of the downstream experiment;
c) Add 20 μl of nuclease free-water to each tube to dissolve the RNA, then place the RNA in a PCR machine, denature it at 95 ° C for 5 min, and immediately place it on ice for later use.
3) Cell lysis
a) discard the medium, wash it 3 times with pre-cooled PBS, and aspirate the supernatant;
b) Add 200 μl of cell lysis buffer A (additional protein triple) to each dish;
c) scrape the cells with a cell scraper, collect them into the EP tube, and triple-frozen in liquid nitrogen;
d) centrifugation, 4 ° C, 3000 rpm, centrifugation for 10 min;
e) Transfer the supernatant to a new EP tube and place on ice; add 200 U/ml RNase Inhibitor;
f) Take about 10-20 μl of supernatant to the new tube as the experimental input group.
4) Magnetic beads (PuriMag G-strep article number PMG015 ) pretreatment
a) vortex mixing beads;
b) Take 400 μl of beads per tube (as in this experiment, one tube of control RNA and one RNA of interest), place on a magnetic stand, and aspirate the supernatant;
c) Wash with 800 μl of 1X binding & washing buffer 3 times and aspirate the supernatant;
5) RNA combined with beads
a) Add 400μl 2X binding & washing buffer to the previous step of the beads;
b) adding 20 μl of RNA to the previous step, and then supplementing 380 μl of DEPC water to a final volume of 800 μl;
c) Slowly rotate at room temperature for 20 min to allow the beads to fully bind to the RNA;
d) placing the tube of the previous step on a magnetic stand and sucking off the supernatant;
e) Wash with 800 μl of 1X binding & washing buffer (containing RNase Inhibitor, 1 U/μl) for 3 times, and aspirate the supernatant;
f) Wash the beads with cell lysis buffer A, aspirate the supernatant, taking care not to let the beads dry.
6) RNA and protein interaction
a) Add 500 μl of cell lysate to each tube of processed bead in the previous step (Step 3); add RNase Inhibitor, 1 U/μl to each tube;
b) Slowly rotate at 4 °C for 2 h to allow the beads to fully bind to the cell lysate;
c) Aspirate the supernatant on a magnetic stand and wash the beads 5 times with 400 μl of freshly prepared cell lysis buffer A (+RNase Inhibitor+ protein triple);
d) Aspirate the supernatant, resuspend the beads with 25 μl of pre-cooled 0.1% SDS solution, add 6.25 μl of 5X protein loading buffer, and boil for 10 min in a 100 ° C metal bath. Immediately on ice for 5 min, then placed in magnetic force. The supernatant was pipetted into a new EP tube to prepare the protein for loading.
7) Detection of protein
Validation of downstream proteins can be continued using western blot or mass spectrometry.
The above is the super detailed cheats of the RNA pull-down technology!
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