throughout the run and prevents alkaline hydrolysis of the
coprecipitated RNA.
4. The optimal dose of UV light is determined by inspection of
the autoradiograms of the RNA-protein complexes separated
on SDS polyacrylamide gels (see Subheadings 3.10 and 3.11).
The optimal size of fragmented RNA ranges between 30 and
300 nucleotides, which corresponds to 15–80 kDa above the
expected molecular weight of the precipitated protein (Fig. 2).
5. Binding of contaminating RNAs to the beads is minimized by
preclearing the cellular lysate with plain beads.
6. Cut off the end of the tips when pipetting beads.
7. Use LoBind tubes (Eppendorf) to avoid unspecific binding of
proteins and nucleic acids to the walls of the tube during
IP. Change tubes during IP to remove residual proteins and
nucleic acids which bind nonspecifically to the walls.
8. In case of high RNase activity in the total extract, the use of
additional RNase inhibitors like vanadyl-ribosyl complex
(VRC) or heparin sodium salt might be necessary.
9. Washing conditions have to be established carefully to reduce
unspecific binding as much as possible but to avoid dissociation
of the specific RNA-protein interactions and of the
RBP-antibody interaction. Stringency of washing buffer can
be changed by adjusting the concentration of NaCl, urea, or
other chaotropic salts, e.g., LiCl.
10. Optimal RNase I conditions are crucial and have to be titrated
for each RBP individually. Therefore, RNase I dilutions ranging from 1:100 to 1:6000 have to be tested. The efficiency of
the RNase treatment can be monitored by visualization of the
RNA-protein complexes via SDS-PAGE and autoradiography
(see Note 3) (Fig. 2).
11. Pipette carefully as LDS easily produces foam.
12. Leave lanes free between the samples.
13. Use different scalpels for the different samples and controls to
avoid cross-contamination.
14. Measure radioactivity on the beads. Above 90% of the radioactivity should be removed after digestion.
Acknowledgments
We greatly appreciate valuable suggestions on the protocol by Drs.
Julian Ko ¨nig, Kathi Zarnack, and Michaela Mu ¨ller Mc-Nicoll. This
work was supported by the DFG (STA 653/6, STA653/9, and
STA653/14).
266
Tino Ko ¨ ster and Dorothee Staiger
coprecipitated RNA.
4. The optimal dose of UV light is determined by inspection of
the autoradiograms of the RNA-protein complexes separated
on SDS polyacrylamide gels (see Subheadings 3.10 and 3.11).
The optimal size of fragmented RNA ranges between 30 and
300 nucleotides, which corresponds to 15–80 kDa above the
expected molecular weight of the precipitated protein (Fig. 2).
5. Binding of contaminating RNAs to the beads is minimized by
preclearing the cellular lysate with plain beads.
6. Cut off the end of the tips when pipetting beads.
7. Use LoBind tubes (Eppendorf) to avoid unspecific binding of
proteins and nucleic acids to the walls of the tube during
IP. Change tubes during IP to remove residual proteins and
nucleic acids which bind nonspecifically to the walls.
8. In case of high RNase activity in the total extract, the use of
additional RNase inhibitors like vanadyl-ribosyl complex
(VRC) or heparin sodium salt might be necessary.
9. Washing conditions have to be established carefully to reduce
unspecific binding as much as possible but to avoid dissociation
of the specific RNA-protein interactions and of the
RBP-antibody interaction. Stringency of washing buffer can
be changed by adjusting the concentration of NaCl, urea, or
other chaotropic salts, e.g., LiCl.
10. Optimal RNase I conditions are crucial and have to be titrated
for each RBP individually. Therefore, RNase I dilutions ranging from 1:100 to 1:6000 have to be tested. The efficiency of
the RNase treatment can be monitored by visualization of the
RNA-protein complexes via SDS-PAGE and autoradiography
(see Note 3) (Fig. 2).
11. Pipette carefully as LDS easily produces foam.
12. Leave lanes free between the samples.
13. Use different scalpels for the different samples and controls to
avoid cross-contamination.
14. Measure radioactivity on the beads. Above 90% of the radioactivity should be removed after digestion.
Acknowledgments
We greatly appreciate valuable suggestions on the protocol by Drs.
Julian Ko ¨nig, Kathi Zarnack, and Michaela Mu ¨ller Mc-Nicoll. This
work was supported by the DFG (STA 653/6, STA653/9, and
STA653/14).
266
Tino Ko ¨ ster and Dorothee Staiger