I am trying to produce polyclonal antibodies against a protein in mice that
I have blotted onto nitrocellulose (there are reasons this animal and
nitrocellulose were choosen). I was wondering if anyone has experience
injecting a mouse with a nitrocellulose prep. The protocols I have tried
were taken from "Antibodies-a laboratory manual" from cold springs harbor
and they include freeze fracturing the nitrocellulose with liq. nitrogen,
dissolving the membrane with acetone or DMSO. None of these seem to work
for me as well as they are described in the procedures. For example, when I
freeze fracture the pieces produced sometimes clog the syringe and I don't
think I'm injecting as much protein as I'd like to. When I dissolve with
acetone and let it evaporate, the nitrocellulose reforms and doesn't go into
solution well. And lastly, when I dissolve it in DMSO, not all the membrane is
dissolved. I don't want to use larger volumes of the chemical to dissolve
it because I can only inject a small volume of sample into a mouse and I'd
like as much of it as possible to be protein.
If anyone has tried any of these or other protocols with any success, I'd
like to hear about them. Also if similar problems were encountered (or if any
ways to circumvent the problems are known), please let me know!
ANy help or insight is appreciated!!!
U. of MD, Microbiology Dept.