RNA Detection

9. Rotating wheel for falcon tubes.


10. Liquid nitrogen.

2.2 Solutions We routinely use distilled water (MilliQ grade) to prepare the
solutions described below.

1. Adherent cell line, such as HEK293, HeLa, U2OS, and MCF-
   7 (ATCC).

( )

XAAAAAA

m/z

Label RNA with

photoactivatable

ribonucleoside

4-thiouridine (4SU)

Oligo(dT) affinity purification

T T T T T T T T T T T T

Mass spectrometry

Determine differential binders

(normalize to “heavy” intensity)

4SU

Covalently crosslink RNA to protein

(UV365 nm irradiation)

Lyse

intensity

AAAAAA

AAAAAA

X

X

X X

4SU

(denaturing, 1% LiDS)

Spike-in SILAC “heavy” lysate

into both samples

AAAAAA AAAAAA

AAAAAA

X

X

X X

m/z

AAAAAA AAAAAA

AAAAAA

X

X

X X

X X

X X

X

X X

X

treated sample untreated sample

treated cells untreated cells

protein-RNA crosslinks

XAAAAAA

XX

AAAAAA

XXX AAAAAA

X

XX

XXAAAAAA

XAAAAAA

XAAAAAA

nuclease digestion

XAAAAAA

XXXXAAAAAA

XAAAAAA

XXXAAAAAA

XAAAAAA

XAAAAAA

nuclease digestion

4SU

SILAC “heavy”

UV crosslink

Lyse

standard culture media (non-SILAC)

Treat cells

intensity

4SU

4SU

4SU

AAA 4SUAAA

Fig. 1Graphical representation of experimental procedure to detect differential protein binding to poly(A)+RNA
upon biological stimulus. In order to be able to accurately normalize the data between two experimental
conditions, we rely upon the usage of “heavy” SILAC lysate obtained from separately cultured “heavy”-labeled
cells. Cell lysates obtained from untreated or treated “light” cells are spiked with the same volume of “heavy”
SILAC lysate, followed by oligo(dT) affinity purification. Protein–mRNA-containing eluates are then nuclease-
treated, concentrated and used in mass spectrometry runs. The measured light peptide intensities can be
normalized to the heavy intensities, which are present due to the spike-in

Isolation and Differential Analysis of RBPs 407