RNA Detection

14. Keep the eluates on ice or store them at 80 C until further
    use. Wash and store oligo(dT) beads in lysis/binding buffer at
    4 C. Reuse them up to three times.

3.3 Analyses of Oligo
(dT) Eluates

1. Save an aliquot of the eluate (100μL) for further RNA analyses
   and store it at 80 C(seeNote 12).


2. To prepare the remaining eluate for mass spectrometry analysis,
   digest RNA by incubation with RNAse I and benzonase. Dilute
   the RNAse I, benzonase and 1 M MgCl 2 1:1000 in the eluate
   to obtain the final concentrations of 100 U/mL, 125 U/mL
   and 1 mM, respectively. Incubate for 1 h at 37C in a water
   bath.


3. Concentrate the eluate by the 15 mL centrifugal filter. Load
   your sample into the filter unit and fill up the filter device with
   concentrating buffer to a total volume of 14 mL. Centrifuge
   the filter unit at 4000gfor 45 min at 4C(seeNote 13).
   Recover the retentate from the filter device (approximately
   150 μL).


4. Analyze the protein sample by silver staining (Fig.2a) and
   Western analysis.


5. Submit the sample for mass spectrometry analysis and analyze
   the data to obtain the identity of enriched RNA-binding pro-
   teins (seeNote 14).

3.4 Detection of
Differences in Binding
Activity Upon a
Biological Stimulus

General consideration: If studying stress responses (e.g., oxidative

stress, ER stress, and DNA damage), it is important to evaluate the

effect of perturbations (4SU treatment, UV exposure, SILAC

media incubation) on stress induction and cell growth, as well as

4SU labeling efficacy (seeNote 15).

1. Grow a population of cells in SILAC heavy media formulation
   and passage them at least six times before proceeding with the
   experiment (seeNote 16).


2. Once the SILAC cells are expanded and around 70–80% con-
   fluent, grow additional two populations of cells in “light” (i.e.,
   non-SILAC) media until 80% confluency.


3. Incubate all three cell populations with 200 μM 4SU for
   6–10 h (seeNote 17).


4. Perform stimulation for one “light” cell population.


5. After the desired incubation, UV-cross-link the cells as
   described in Subheading3.1 (seeNote 18).


6. Immediately harvest UV-exposed cells by scraping them off in
   5 mL ice-cold PBS using a rubber policeman. Transfer cell
   suspensions into 50-mL falcon tubes and pellet them by centri-
   fugation (400g,4C, 5 min). Wash the cell pellet once with

Isolation and Differential Analysis of RBPs 411