Enrichment and identification method of RNA-protein compound
A protein complex and enrichment technology, applied in the field of analytical chemistry, can solve the problems of high cost of reagents, inability to apply tissue samples, limited metabolic labeling efficiency, etc., and achieve the effect of reducing steric hindrance, comprehensive information, and improving enrichment efficiency
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Embodiment 1
[0044] Example 1. Evaluation of the effect of PB reagent on enriching RNA-protein complexes in cells
[0045] When the HeLa cells cultured in a 15-cm petri dish had a plate coverage rate of approximately 80%, the culture medium in the petri dish was removed, and the cells were rinsed with PBS 3 times (5 mL each time). After blotting the solution in the petri dish, place the petri dish on ice for UV cross-linking. The ultraviolet wavelength is 254nm, the power is 40W, and the irradiation time is 1min. Add ice PBS solution, use a cell scraper to collect the cells in a 2mL RNase-free centrifuge tube, centrifuge at 1,000g for 3min, and discard the supernatant. After adding 250μL of Lysis Solution I[PBS,0.5%(w / v)SDS,EDTA-free protease inhibitormixture(Thermo),ribonucleosidevanadyl complex(NEB)], place the centrifuge tube on ice and use a thin needle (~0.7 mm) the syringe is fully homogenized and allowed to stand for 20 min. Add 1mL lysate II (PBS, 0.1% triton-100, EDTA-free proteas...
Embodiment 2
[0048] Example 2. RNA sequencing analysis of RNA-protein complexes in PB reagent enriched cells
[0049] Using the same experimental conditions as in Example 1, after the RNA-protein complex was captured by magnetic beads, the supernatant was discarded by magnetic separation. The magnetic beads were used in turn with 200 μL 0.2% (w / v) SDS in PBS and 200 μL respectively. Wash each with 8M urea in PBS solution and 200μL PBS solution. Add 400μL of eluent [12.5mM biotin, 75mM NaCl, 7.5mM Tris·HCl (pH 7.5), 1.5mM EDTA, 0.15% SDS, 0.075% sodium lauryl sarcosinate and 0.02% sodium deoxycholate], Vortex for 20 minutes (800 rpm) at room temperature. Then vortex at 65°C for 10 minutes (800 rpm) on a thermostatic vortex. After eluting the beads once, add a new 400μl eluate and repeat the above elution step. The two eluates were combined to obtain a total of 800 μl of solution. Add 2mg / mL proteinase K and digest at 55°C for 1 hour. After the protein in the complex is completely degraded...
Embodiment 3
[0051] Example 3. Proteome mass spectrometry analysis of RNA-protein complexes in PB reagent enriched cells
[0052] The experimental process is as Figure 5 Shown.
[0053] Experimental group: Using the same experimental conditions as in Example 1, after obtaining the purified RNA-protein complex by magnetic bead capture, the supernatant was discarded by magnetic separation, and the magnetic beads were respectively used 200μL 0.2%(w / v) SDS in PBS solution, 200μL 8M urea in PBS solution, 200μL PBS solution, 200μL 50mM NH 4 HCO 3 Each wash once. Discard the supernatant by magnetic separation, add 20μL 0.01μg / μL RNase A (50mM NH 4 HCO 3 ), incubate at 37°C for 1h. Then carry out trypsin digestion, the specific operation is as follows: add 10mM (final concentration) dithiothreitol to 56℃ water bath to reduce for 1 hour, then add 50mM (final concentration) iodoacetamide and place in the dark for 30min for alkylation treatment , Take the denatured protein and add 0.02μg trypsin, place...
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