protein extraction of embryos for westerns

[Nancy L. Hardison]

i have been attempting to quantitate the amount of gfp in transgenic
zebrafish by western blotting.

has anyone tried to do this before with zebrafish?

i have been using the protocol for harvesting the proteins from the 
zebrafish book and from the zebrafish science monitor.  i am 
deyolking the embryos in ringers plus edta and pmsf and 
homogenizing in their sds sample buffer.  my results have been 
varied but i consistently get a giant smear that runs between 
55,000 and 75,000 and usually, the gfp band at 27,000. the primary 
is molecular probes A-11122 IgG rabbit anti-gfp (at 1:1500 either 2 
hrs room temperature or 4 degrees overnight) and the secondary is 
jackson immuno anti-rabbit alk phos (1:10,000 one hour room 
temperature) and i am using tropix chemiluminescent detection 
system.  i have run a secondary antibody with no primary and have 
shown that the secondary is clean and does not bind to the 
zebrafish proteins (and thus the chemiluminescent substrate isn't 
reacting to the zebrafish proteins either.)  i am baffled.  i am 
assuming the primary is sticking to something.  i vaguely 
remember doing westerns on tissue culture cells where i would 
incubate the cells in a lysis buffer.  could i not be homogenizing 
the cells properly?  

if anyone has any ideas, advice, or protocols that would be helpful, i
would GREATLY appreciate it.

thank you very much

nancy hardison
email:  nancy.hardison at duke.edu
phone:  919-684-6744
fax:  919-684-8735



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