- Place the frozen tissues into the Dounce homogenizers con-
taining 400μL of ice-cold RO-lysis buffer (10 w/v % tissues in
RO-lysis buffer). - Centrifuge the lysate at 16,000gat 4C for 10 min. During
this step, prepare the rabbit reticulocyte lysate (RRL) as follow-
ingsteps 4– 6. - Add the 8μL harringtonine (100μg/mL stock solution) and
4 μL 4E1RCat (5 mM stock solution) to 188μL RRL, and mix
gently. For the “no run-off” control, add 8μL cycloheximide
to the mix (seeNote 10) - Preincubate RRL at 30C for 5 min
- Transfer the supernatant (step 3) to the tube and mix it gently.
- Split the lysate (step 6) into two tubes. One of two tubes can be
used for the “no run-off” control. - Add 200μL of RRL mix (step 4) to 200μL of the lysate (step
7 ), and incubate at 30C for 30 min. - Add 800μL ice-cold CHX-lysis buffer to the run-off reaction
and incubate the tube on ice for 5 min to stop the translation
elongation. - Proceed to immunoprecipitation, following thesteps 5– 10 of
the Subheading3.3 and then Subheading3.4.
4 Notes
- Make sure that no cells in the target tissue (which contains axon
terminals of interest and in this case is the rostral half of the
superior colliculus) express tagged ribosomes. Histological
assays using any Cre reporter mice (e.g., Rosa26-Stop-LoxP-
EGFP) is a good start. Confirm this in the RiboTagCre
mouse to be studied using HA immunohistochemical detec-
tion. PCR-based detection of chromosomal DNA that under-
went Cre-mediated recombination is a more sensitive method.
The “normal” RiboTag allele and “recombined” RiboTag
allele can be distinguished by PCR (Fig.2)[5]. The target
tissue should not contain a detectible level of the recombined
RiboTagallele. Use the following primers to detect normal
and recombined RiboTag alleles: RiboTag fwd, 5^0 -
GGGAGGCTTGCTGGATATG-3^0 , and HA rev, 5^0 -ACATCG-
TATGGGTATAGATCC-3^0 (Fig.2). - Wear safety glasses or a face shield when using liquid nitrogen.
- Prepare all solutions just before the experiment.
Isolation of In Vivo Axonal Translatome 91