protein extraction zebrafish embryo

Daniel Prieto via methods%40net.bio.net (by dprieto from fcien.edu.uy)
Thu Jul 22 15:56:43 EST 2010


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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