- q-PCR and/or Agilent Bioanalyzer.
- TurboDNase with high salt tolerance.
- Glass dounce homogenizer, 2 mL size.
- UV cross-linker with 254 nm wavelength bulbs (e.g.,
Spectrolinker). - Vacuum lyophilizer.
- Streptavidin-coated magnetic beads (e.g., Life Technologies
Dynabeads). - Protease inhibitor cocktail set III, EDTA free.
- Benzonase nuclease.
- Detergent removal columns (e.g., Life Technologies HiPPR).
Fig. 4Example of SILAC ratio plot for proteins from RAP-MS captures. Captures
from more than one RNA target can be mixed and quantitated by mass spec-
trometry. RAP-MS captures for U1 and 18S were performed in both heavy and
light lysates and resulting proteins were mixed together. Proteins identified in
two replicates with label swap (18S light label vs. U1 heavy label, 18S heavy
label vs. U1 light label) are plotted by their log 2 SILAC ratio. Proteins that
replicate in label-swap captures are high-confidence interactors for the target
RNA molecule. Known contaminants (keratins, trypsin, benzonase, and strepta-
vidin) are always purified in the light labeled sample and can be excluded from
the final list of interactors. Adapted from [4]
Identification of Direct RNA Binding Proteins Using RAP-MS 477