- Liquid nitrogen in a container (seeNote 2).
- Dissection tools: forceps, fine scissors, and springbow dissect-
ing scissors.
2.2 Lysis and
Immunoprecipitation
Prepare all solutions using RNase-free water, buffers, and labware.- A Dounce homogenizer.
- CHX-Lysis buffer: 20 mM HEPES–KOH, 5 mM MgCl 2 ,
150 mM KCl, 1 mM DTT, 1 v/v % NP40, 200 U/mL
SUPERase In, 100 μg/mL cycloheximide, and Complete
EDTA-free Protease Inhibitor Cocktail (seeNote 3). - Wash buffer: 20 mM HEPES–KOH, 5 mM MgCl 2 , 350 mM
KCl, 1 mM DTT, 1 v/v % NP40, and 100 μg/mL
cycloheximide. - Polyclonal anti-HA antibody (Abcam, ab9110).
- Magnetic Protein G beads (e.g., Thermo Fisher Scientific
Dynabeads). - A rotator for Eppendorf tubes in a cold room (4C).
- Magnetic Eppendorf stand (e.g., Thermo Fisher Scientific
DynaMag). - RNeasy mini kit (Qiagen).
- 2-mercaptoethanol.
2.3 Run-Off
Translation
- A Dounce homogenizer.
- RO-lysis buffer: 20 mM HEPES-KOH, 5 mM MgCl 2 ,150mM
KCl, 1 mM DTT, 200 U/mL SUPERase In, Complete EDTA-
free Protease Inhibitor Cocktail (Roche, cOmplete), 1% volume
of amino acid mix (leucine), and 1% volume of amino acid mix
(methionine) (amino acids mixes are included in Flexi Rabbit
Reticulocyte Lysate System (Promega, L4540). - Flexi Rabbit Reticulocyte Lysate System (Promega, L4540).
- 100μg/mL Harringtonine stock solution.
- 5 mM 4E1RCat stock solution.
- 5 mg/mL cycloheximide.
- Wash buffer: 20 mM HEPES-KOH, 5 mM MgCl 2 , 350 mM
KCl, 1 mM DTT, 1% NP40, and 100μg/mL cycloheximide
(seeNote 4).
3 Methods
3.1 Mouse Breeding 1. A Pax6-alpha-Cre hemizygotic male mouse is mated with a
RiboTag homozygotic female mouse. Each litter is expected
to have roughly equal numbers of Cre-positive (the experimen-
tal group) and Cre-negative (the negative control) animals.
88 Toshiaki Shigeoka et al.