2. Spin down (5 min at 958 Â g in a microcentrifuge) and transfer
supernatant to a new tube.
3. Concentrate in the SpeedVac for >2 h at room temperature.
Check the samples approximately once every hour until all the
liquid has evaporated, leaving behind a solid powder at the
bottom of the tube (see Note 20).
4. Store at À20
C and submit for liquid chromatography-mass
spectrometry.
3.3.4 Mass Spectrometry
RNA affinity purification should be followed by liquid
chromatography-mass spectrometry to identify enriched protein
candidates. The liquid chromatography-mass spectrometry
(LC-MS) technique uses liquid chromatography principles to separate peptides and then identifies them with mass spectrometry.
For well-characterized model organisms, label-free methods such
as the iBAQ system can be used in the mass spectrometry portion
[15]. Other mass spectrometry methods include stable isotope
labeling methods [16] or selective reaction monitoring
[17]. Based on the analysis of protein sequence similarity and
RNA-binding motif similarity, proteins with >70% amino acid
sequence similarity are likely to have very similar RNA sequence
specificity [18], so users should expect several candidate proteins to
be identified after mass spectrometry analysis.
3.3.5 Further Validation
Once mass spectrometry analysis has identified possible RBP candidates of interest, further possible validation experiments include
western blots, knockout/knockdown experiments, CLIP-seq to
validate that the protein interacts with the motif that was used to
pull it down, etc.
4 Notes
1. RNA oligos can be purchased from services such as IDT. In
general, we recommend probe sequences of >20 bases. The
oligo comes as lyophilized content and will need to be reconstituted with ribonuclease (RNase)-free water. To make up
100 μM of the RNA probe, add in x μL of nuclease-free water
where x is 10∗ the concentration of the oligo in nmoles. For
example, if the probe is 89.4 nmol, add 894 μL of RNase-free
water to reconstitute to 100 μM. Properly stored at À20
C,
probes can be stable for up to 2 years. For longer term storage,
probes should be aliquoted and kept at À80
C.
2. Sodium m-periodate is light sensitive and should be stored in
the dark and the solution made fresh daily.
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Mengge Shan and Brian D. Gregory
supernatant to a new tube.
3. Concentrate in the SpeedVac for >2 h at room temperature.
Check the samples approximately once every hour until all the
liquid has evaporated, leaving behind a solid powder at the
bottom of the tube (see Note 20).
4. Store at À20
C and submit for liquid chromatography-mass
spectrometry.
3.3.4 Mass Spectrometry
RNA affinity purification should be followed by liquid
chromatography-mass spectrometry to identify enriched protein
candidates. The liquid chromatography-mass spectrometry
(LC-MS) technique uses liquid chromatography principles to separate peptides and then identifies them with mass spectrometry.
For well-characterized model organisms, label-free methods such
as the iBAQ system can be used in the mass spectrometry portion
[15]. Other mass spectrometry methods include stable isotope
labeling methods [16] or selective reaction monitoring
[17]. Based on the analysis of protein sequence similarity and
RNA-binding motif similarity, proteins with >70% amino acid
sequence similarity are likely to have very similar RNA sequence
specificity [18], so users should expect several candidate proteins to
be identified after mass spectrometry analysis.
3.3.5 Further Validation
Once mass spectrometry analysis has identified possible RBP candidates of interest, further possible validation experiments include
western blots, knockout/knockdown experiments, CLIP-seq to
validate that the protein interacts with the motif that was used to
pull it down, etc.
4 Notes
1. RNA oligos can be purchased from services such as IDT. In
general, we recommend probe sequences of >20 bases. The
oligo comes as lyophilized content and will need to be reconstituted with ribonuclease (RNase)-free water. To make up
100 μM of the RNA probe, add in x μL of nuclease-free water
where x is 10∗ the concentration of the oligo in nmoles. For
example, if the probe is 89.4 nmol, add 894 μL of RNase-free
water to reconstitute to 100 μM. Properly stored at À20
C,
probes can be stable for up to 2 years. For longer term storage,
probes should be aliquoted and kept at À80
C.
2. Sodium m-periodate is light sensitive and should be stored in
the dark and the solution made fresh daily.
250
Mengge Shan and Brian D. Gregory