- Different RBPs behave differently during or upon lysis. For
every protein, lysis conditions have to be optimized. Impor-
tantly, Mili and Steitz raised the important experimental issue
that there might be reassociation of molecules, especially for
RBPs, after cell lysis [7]. - OptiPrep gradients have to be optimized for each RNP of
choice. The migration of the respective RBP into the gradient
strongly depends on the granule density that is influenced by
the number of proteins and/or RNAs embedded in those
particles. - Check gradient fractions on Western blot. Generally, Stau2
containing RNA granules are expected to accumulate in frac-
tion 4–6 while Btz granules are predominately found in frac-
tion 5–7 [6]. - We recommend to compare Western blots with Coomassie gels
of the respective fractions. Pool those fractions that show a
signal in the Western blot for your protein of interest and less
staining intensity of the total protein. - RNase A + T1 requires Mg2+to work. For RNA extraction, add
TRIzol to the beads after washing. For RNA isolation, it is
recommended to increase the stringency of washing steps by
increasing NP-40 concentration. - The efficiency of RNA digestion strongly depends on the cho-
sen RNase. This step has to be optimized. Keep in mind, some
RNases cut only single stranded or double stranded RNA while
others cut both. - Cross-linked beads can be reused several times. Importantly,
the yield of proteins in the elution fraction is decreasing with
frequency of usage. - For analysis of protein interactors or RNA targets, we used the
PIS from the same rabbit as negative control. For analytical
immunoprecipitation, proteins eluted from antibody and PIS
beads are precipitated by trichloroacetic acid or methanol chlo-
roform extraction [8].
References
- Kiebler MA, Bassell GJ (2006) Neuronal RNA
granules: movers and makers. Neuron
51:685–690. doi:10.1016/j.neuron.2006.08.
021 - Jung H, Gkogkas CG, Sonenberg N, Holt CE
(2014) Remote control of gene function by local
translation. Cell 157:26–40. doi:10.1016/j.cell.
2014.03.005
3. Doyle M, Kiebler MA (2012) A zipcode
unzipped. Genes Dev 26:110–113. doi:10.
1101/gad.184945.111
4. Buxbaum AR, Haimovich G, Singer RH (2014)
In the right place at the right time: visualizing
and understanding mRNA localization. Nat Rev
Mol Cell Biol 16:95–109. doi:10.1038/
nrm3918
5. Heraud-Farlow JE, Sharangdhar T, Li X et al
(2013) Staufen2 regulates neuronal target
Isolation of RNA Granules 425