15. There should be one 15 mL tube with beads for each probe
sample. For example, if there are four samples total, then there
should be four tubes with 100 μL of beads in each instead of
one tube with 400 μL of beads.
16. The resuspended beads can be stored at 4
C until needed.
17. The agarose beads can be difficult to visualize. Users can use a
pipette and remove a little bit less than the amount of liquid
put in during previous steps. For example, if the previous step
added in 1 mL of liquid, remove 900 μL. Use a new pipette tip
for each tube to minimize cross-contamination. Tilting the
tube slightly can also help visualize the concentration of
beads at the bottom of the tube.
18. Depending on the concentration of the lysate, the binding
buffer may not be exactly 1.5 mL. Users can make up the
difference with additional RNase-free water.
19. Some mass spectrometry protocols will recommend adding a
reduction and alkylating step at this point to improve the
quality of downstream mass spectrometry data. If so, instead
of resuspending in 100 μL 100 mM NH 4 HCO 3 , resuspend in
100 μL of 10 mM DTT in 100 mM NH 4 HCO 3 and then
incubate for 1 h at 56
C. After cooling the sample to room
temperature, add iodoacetamide to a final concentration of
50 mM and incubate in the dark for 45 min. Iodoacetamide
is unstable and light sensitive, which means 1 M stock needs to
be made fresh every day and kept in the dark for as much time
as possible when performing the experiments, especially once it
is in solution.
20. Depending on the number of samples and the room humidity,
concentrating the samples can take longer than 2 h. As long as
the vacuum is operating normally and the liquid is evaporating,
simply monitor the samples until the supernatant has
evaporated.
Acknowledgments
The authors would like to thank members of the Gregory lab both
past and present for helpful discussions. This work was funded by
NSF grants MCB-1243947, MCB-1623887, and IOS-1444490 to
B.D.G.
References
1. Glisovic T, Bachorik JL, Yong J, Dreyfuss G
(2008) RNA-binding proteins and posttranscriptional gene regulation. FEBS Lett
582:1977–1986
2. Lukong KE, Chang K, Khanjian EW, Richard S
(2008) RNA-binding proteins in human
genetics disease. Trends Genet 24:416–425
252
Mengge Shan and Brian D. Gregory
sample. For example, if there are four samples total, then there
should be four tubes with 100 μL of beads in each instead of
one tube with 400 μL of beads.
16. The resuspended beads can be stored at 4
C until needed.
17. The agarose beads can be difficult to visualize. Users can use a
pipette and remove a little bit less than the amount of liquid
put in during previous steps. For example, if the previous step
added in 1 mL of liquid, remove 900 μL. Use a new pipette tip
for each tube to minimize cross-contamination. Tilting the
tube slightly can also help visualize the concentration of
beads at the bottom of the tube.
18. Depending on the concentration of the lysate, the binding
buffer may not be exactly 1.5 mL. Users can make up the
difference with additional RNase-free water.
19. Some mass spectrometry protocols will recommend adding a
reduction and alkylating step at this point to improve the
quality of downstream mass spectrometry data. If so, instead
of resuspending in 100 μL 100 mM NH 4 HCO 3 , resuspend in
100 μL of 10 mM DTT in 100 mM NH 4 HCO 3 and then
incubate for 1 h at 56
C. After cooling the sample to room
temperature, add iodoacetamide to a final concentration of
50 mM and incubate in the dark for 45 min. Iodoacetamide
is unstable and light sensitive, which means 1 M stock needs to
be made fresh every day and kept in the dark for as much time
as possible when performing the experiments, especially once it
is in solution.
20. Depending on the number of samples and the room humidity,
concentrating the samples can take longer than 2 h. As long as
the vacuum is operating normally and the liquid is evaporating,
simply monitor the samples until the supernatant has
evaporated.
Acknowledgments
The authors would like to thank members of the Gregory lab both
past and present for helpful discussions. This work was funded by
NSF grants MCB-1243947, MCB-1623887, and IOS-1444490 to
B.D.G.
References
1. Glisovic T, Bachorik JL, Yong J, Dreyfuss G
(2008) RNA-binding proteins and posttranscriptional gene regulation. FEBS Lett
582:1977–1986
2. Lukong KE, Chang K, Khanjian EW, Richard S
(2008) RNA-binding proteins in human
genetics disease. Trends Genet 24:416–425
252
Mengge Shan and Brian D. Gregory