Chapter 9 - Molecular Methods
Extraction of Proteins from Zebrafish Embryos for SDS-Gel Analysis
(Source: M. Westerfield)
1. Remove embryos from their chorions by immersing them in 1 mg/ml
pronase for 5 min, RT, in a petri dish. Embryos younger than 24 h
require shorter exposure to pronase (see
Removing Embryos from their Chorions, Chapter 4).
2. Transfer them to a clean beaker filled with 10% Hank's saline and rinse
several times with clean 10% Hanks.
3. Triturate gently with a pasteur pipette to expel the embryos from their
chorions, if necessary.
4. Transfer embryos into microcentrifuge tubes (1.5 ml Eppendorf). Remove
as much water as possible with a pipette.
5. Add cold SDS sample buffer (63 mM Tris-HCl pH 6.8, 10% glycerol,
5% ß-mercaptoethanol, 3.5% sodium dodecyl sulfate; see below or RECIPES, Chapter 10) at 1 µl per fish.
6. Homogenize with pestle. Add another 1 µl of SDS sample buffer
per fish to rinse pestle, homogenize a bit more.
7. Immerse tube in boiling water for 1 min.
8. Spin in a microcentrifuge for 5 min. Collect supernatant and apply to gel.
Alternatively, samples can be frozen at this point (-70°C) for later
analysis.
SDS sample buffer
0.63 ml 1M Tris-HCl, pH 6.8
1.0 ml glycerol
0.5 ml ¸-mercaptoethanol
1.75 ml 20% SDS
6.12 ml H2O
(10 ml total)
Store at -20°C in aliquots.
The Zebrafish Book
