This material is from the 4th edition of The Zebrafish Book. The 5th edition is available in print and within the ZFIN Protocol Wiki.
CHAPTER 9 - MOLECULAR METHODS
Western Blots of Zebrafish Embryos
(Source: S. O'Shea and M. Westerfield)
Dechorionating & deyolking
1. Remove embryos from their chorions in
batches of ~100 by placing in 1 mg/ml of
pronase and swirling occasionally (5-10
minutes for 24 h embryos, 10-20 minutes
for 3 day embryos). Finish dechorionation
by gentle trituration using a Pasteur pipette.
The chorions float and can be decanted.
Rinse three times in cold Ringer's
solution. (See also Removing Embryos
from their Chorions, Chapter 4).
2. Transfer the embryos
to cold Ringer's with EDTA and PMSF. Remove
with a glass pipette that has been drawn
out to have a tip diameter approximately
the size of the
3. Transfer the dechorionated,
deyolked embryos to fresh, cold Ringer's
solution and rinse twice.
4. Embryos can be frozen in liquid nitrogen
and stored at -70°C at this point by
transferring to a microcentrifuge tube and
removing as much liquid as
5 mg/ml pronase diluted to 1 mg/ml in
Stock - 100 mM
phenylmethylsulfonylfluoride in isopropanol. Immediately
before use, add 30 µl of stock/10 ml Ringer's (final
conc. 0.3 mM PMSF).
Stock - 10 mM EDTA, pH 7.0. Add 1 ml
of stock/10 ml Ringer's (final conc. 1 mM
Preparation of gel sample
1. Remove from freezer and thaw the frozen,
2. Microfuge for 1-2 minutes to
3. Remove excess liquid.
4. Add 150-200 µl SDS sample buffer
(for example, ~50-100 3 day embryos or
100-150 24 h embryos; this will yield
enough for a 1.5 cm curtain or four 0.4 cm
lanes of about 25-30 µl
5. Homogenize with microfuge pestle until
uniform in consistency.
6. Repeat step 5 until sample is no longer
7. Boil 5 minutes in a water
8. Microfuge, 1-2 min-
9. Transfer supernatant to a new microfuge
tube. Discard pellet or add more sample
buffer and homogenize again if significant
10. Freeze at -70°C or run immediately
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.
For cytoskeleton/extracellular matrix preps:
Add an extraction step between steps 3 and 4 (above).
1. Homogenize dechorionated, deyolked
embryos in extraction
2. Incubate overnight at
3. Centrifuge 20 min at 5000 x g.
Protein extraction buffer
10 mM Tris, pH 7.4
2% Triton-X 100
1 mM PMSF
1 mM aprotinin
1 mM leupeptin
1 mM trypsin inhibitor
Load, run and transfer gel
Follow methods of Towbin, H., T. Staehelin, and J. Gordon (1979) Electrophoretic transfer from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA76: 4350-4354.
Immunoblotting (using PBDF blotting paper)
1. After the antigen is blotted, immerse the
membrane at a 45° angle, into the
blocking solution. Gently agitate for 60 min
at RT or overnight at
2. Decant the blocking solution and add TTBS
to the membrane. Wash for 10 min with
gentle agitation at RT.
3. Decant the TTBS and add the primary
antibody diluted in 1% dried milk in TTBS.
Incubate 4 hr at RT (or overnight at
4°C) with gentle agitation.
4. Remove the unbound 1° antibody by
washing twice for 5 min each in
5. Add alkaline-phosphatase conjugated
2° antibody solution (diluted 1:3000
in 1% dried milk in TTBS). Incubate 1-2 hr
at RT with gentle
6. Wash membrane twice for 5 min each in
TTBS and then, just prior to color develop-
ment, once for 5 min in TBS to remove the
7. Immerse membrane in color development
solution. Proteins present at 100 ng or
greater will immediately become visible as
purple bands. Lower amounts will take
longer, but should be visible within 30 min.
Staining can continue up to 4
8. Rinse membrane four times for 5 min each
3% dried milk in TBS
20 mM Tris, pH 7.5
500 mM NaCl
20 mM Tris, pH 7.5
500 mM NaCl
Western Blot Color Development
66 µl nitroblue tetrazolium (NBT) stock
33 µl 5-bromo-4-chloro-3-indolyl
10 ml color development
Both NBT and BCIP stocks are 50 mg/ml in
dimethylformamide. NBT stock is made
by suspending 50 mg NBT in 700 ml
dimethylformamide. Vortex. Add 300
µl distilled water to dissolve.
Store both stocks at 4°C in