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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.
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