- Analyze the oligo(dT) eluates as described in Subheading3.3.
Representative results are shown in Fig.2b. - Submit the sample for mass spectrometry analysis and analyze
the data to obtain RNA-binding proteins that show differential
binding upon stimulation (seeNotes 21and 22 ).
4 Notes
- 4SU incorporation into RNA may be easily followed by dot
blot analysis as described elsewhere [23]. Determine the con-
ditions of incubations that give you a similar signal as 4SU-
labeled RNA extracted from HEK293/HeLa/MCF-7 cells
that were labeled for 6–10 h in the presence of 100–200μM
4SU. - It is critical to remove the culture medium to allow the maximal
penetration of UV light into the cells. Five 15-cm culture
dishes will fit into the Stratalinker and can be UV irradiated at
the same time. - We typically lyse one cell pellet obtained from an 80–90%
confluent 15-cm culture dish in 1–3 mL of lysis/binding
buffer, depending on cell type. For HEK293 cells the amount
of poly(A)+RNA obtained from one 15-cm culture dish is
high, requiring 3 mL of lysis buffer. Since the amount obtained
from MCF-7 cells is lower, we use 1 mL for lysis. In the
experiments shown in Fig.2b, we used 60 dishes per sample
and lysed the cells in 60 mL of lysis/binding buffer. Note that
such a high amount of starting material can yield 50–150μgof
eluted proteins, and this can be beneficial when a high number
of quantified proteins is needed. However, starting material
may also be decreased to about 5–10 dishes per sample (yield
0.5–5μg) provided the mass spectrometry experiments are
carried out with the current state-of-the art sensitive MS
equipment. - Due to the presence of strong detergent and high salt concen-
tration, the lysate will be very viscous. Viscous material nor-
mally floats at the top of the solution. For efficacy of oligo(dT)
affinity purification it is critical to decrease viscosity and remove
insoluble material. Typically, we pass the lysate four times
through a 23-G needle, followed by 2 passes through a 26-G
needle. Note that it takes around 20–30 min per sample to
perform this step. Be careful to keep the lysate cold at all times.
If insoluble/viscous material is still visible at the top after this
step, try to remove it by pipetting the bottom layer of the
solution into a new precooled 50-mL falcon tube thus
Isolation and Differential Analysis of RBPs 413