We're going to be making some embryo powder for antibody preabsorption
for in situ hybridization. Pretty much as per this protocol: [Only registered users see links. ]
We're wondering whether it's ok to pulverize the embryos in liquid nitrogen
before homogenization in PBS. I'm not sure our homogenizer would be able
to handle whole embryos. That's not going to affect the antigenicity of the
embryo powder, is it?
In article <[Only registered users see links. ].net >, Ed Siefker <[Only registered users see links. ]> wrote:
That would be totally fine.
I have one problem with the protocol though:
Some/many proteins from aceton powder redissolve when
you hydrate it back. So they will end up in your antibody solution
and the antibody against them won't be preabsorbed at all.
Although this will provide some competition with the
antigens in situ, there still will be, inevitably, some
If I were doing it, I would make a who protein extract from
the embryo and coupled the total soluble protein fraction
to a CNBr-activated agarose. Then preabsorb with aceton powder
thoroughly *washed* in buffer (this removes Ab against
insoluble antigens), after which incubating the Ab with the
immobilized total protein will remove Ab recognizing soluble
antigens. More work but better results.