- Resuspend beads in 60μL RLT Buffer (3original sample
volume). - Transfer beads in RLT to 20μL RNA sample and mix well.
- Add 120μL of 100% ethanol (6original sample volume).
Wait for 2 min for sample to bind beads. - Wash beads two times with 150μL of 70% ethanol.
- Remove supernatant and allow to air-dry for approximately
5 min. - Elute RNA in water or desired buffer. In this case, elute RNA
samples by adding 26μLof1TURBO DNase Buffer (dilute
from 10stock supplied by manufacturer). - Leave beads in tube. Perform DNase treatment to remove
background DNA by adding 1μL of murine RNase inhibitor
and 3 μL of TURBO DNase to each sample (30μL total
reaction volume). Incubate for 20 min at 37C. - Perform a second SILANE cleanup using beads already in the
tube: - Add 90μL RLT Buffer to each 30μL sample.
- Add 180μL of 100% ethanol and mix well.
- Wait 2 min for sample to bind beads.
- Wash beads two times with 70% ethanol.
- Remove supernatant and allow beads to air dry approximately
5 min. - Elute in 10μL of UltraPure water.
- Analyze RNA samples using Agilent Bioanalyzer or by RT-
qPCR (seeNote 9).
3.9 Mass
Spectrometry of
Captured Proteins
3.9.1 Protein
Precipitation
- Add 10% final concentration of TCA to protein elution sample.
Incubate at 4C overnight. - Centrifuge at 16,000gfor 30 min to pellet protein.
- Remove supernatant and replace with 1 mL of cold acetone.
- Centrifuge at 16,000gfor 15 min.
- Remove supernatant and allow pellet to dry in open tube in
fume hood or on bench. Store protein elution samples at
20 C.
3.9.2 In-Solution Digest
of Protein Samples for
Mass Spectrometry
- Resuspend protein elution sample in 40μL of freshly prepared
8 M urea dissolved in 100 mM Tris–HCl pH 8.5. - Add 3 mM TCEP and incubate 20 min at room temperature.
- Add 11 mM freshly prepared iodoacetamide and incubate for
15 min at room temperature in the dark.
Identification of Direct RNA Binding Proteins Using RAP-MS 483