Cool! That sounds pretty similar to the protocol on the Zebrafish Book
with some good tips. I'll check your protocol.
Thanks a lot Pow!
>>
Daniel, I made some ZF protein extracts to detect a 110 kDa protein. The
protocol is listed in this paper:
Amyloid precursor protein is required for convergent-extension movements
during Zebrafish development.
<http://www.ncbi.nlm.nih.gov/pubmed/19664615> Joshi P, Liang JO, DiMonte
K, Sullivan J, Pimplikar SW. Dev Biol. 2009 Nov 1;335(1):1-11. Epub 2009
Aug 4.
I believe I used some kind of homogenization in SDS sample buffer,
followed by boiling, and passing through a 26 guage injection needle
5-10 times, followed by centrifugation at high-speed (13000 rpm on a
table top centrifuge). However, we were using embryos from 24 dpf to 72
dpf; anything earlier would require careful removal of the yolk sac
(which you say you are doing manually). The other thing to consider is
using low percentage gels or even gradient gels.
best,
Pow
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