volume of water. Note that due to denaturation some proteins
cannot be effectively resuspended after TCA precipitation.
- After receiving the dataset, compare the intensity values against
those obtained from the non-cross-linked control sample.
Compare log2-fold enrichments and rank the proteins accord-
ingly. To validate novel RNA-binding protein candidates we
stably or transiently express FLAG/HA-tagged version of pro-
teins of interest in cells, which are labeled for 6–10 h with
100–200μM 4SU. After cross-linking the cells with exposure
to 365 nm UV light and the energy of 0.15 J/cm^2 we follow
Subheading3.3,steps 2– 7 of a recently published PAR-CLIP
protocol [24] to examine the formation of protein–RNA com-
plexes which can be detected by radiolabeling RNA in these
complexes.
- Use a biological marker that should be induced upon stimulus
and evaluate the effect of perturbations that are applied to both
unstimulated and stimulated cells, i.e., 4SU treatment, UV
exposure and presence of SILAC media components. For
example, in the case of DNA damage induction, a general
marker for double strand break formation is phosphorylation
of histoneγ-H2AX. Quantity ofγ-H2AX can be followed by
Western analysis or immunofluorescence microscopy.
- In order to prevent the usage of large amounts of SILAC heavy
culture media, grow the cells in small culture dishes for the
initial passages. Since cells generally grow slower in SILAC
media, we do not recommend diluting them lower than three-
fold during passaging. Incorporation of “heavy” amino acids
into proteins may be tested by performing a whole proteome
analysis of nonlabeled/“light” and “heavy” cells. For success-
ful incorporation, one would expect the distribution of SILAC
heavy-to-light ratios to be centered at 1. Although we have not
observed SILAC incorporation to be a critical issue in
HEK293, HeLa, and MCF-7 cells, it may be of great impor-
tance to some specialized cell cultures.
- SILAC media may significantly affect the efficacy of RNA
metabolic labeling by 4SU. Perform optimizations as described
inNote 1.
- If you have many culture dishes for treated cells, make sure that
the incubation time will be the same for all of them by stimu-
lating them at slightly delayed time points. We typically com-
plete the cross-linking and harvesting of five 15-cm dishes in
5–7 min.
- It is a good idea to collect a separate aliquot of cells (1 mL) at
this point to test if the biological marker is present in treated
but not in untreated cells before proceeding with the large-
scale experiment.
Isolation and Differential Analysis of RBPs 415
