protein extraction of embryos for westerns
Nancy L. Hardison
nancy.hardison at duke.edu
Wed Oct 4 14:12:24 EST 2000
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
email: nancy.hardison at duke.edu
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