Western: Mouse antibody on mouse tissue homogenates - (Apr/02/2011 )
Im a newbie and Ive been trying a western on some mouse tissue homogenates. Unfortunately, the only good monoclonal antibody there is, has been raised in mice! Whenever I try a western on these samples, I get a lot(!) of bands! Ive tried incubating the primary and secondary in milk, reducing both the primary and secondary concentrations, but I still see all the non-specific bands! The antibody works perfect on all the invitro samples derived from mouse cells. It also worked fine on the non-mouse tissue homogenates. Apparently, I was going through a post-docs notes and she managed to successfully try the same antibody on mouse samples (I don't know how)!! I've emailed her, but am still waiting for a reply.
Can someone help me.....how do I get some specific bands with a mouse antibody on mouse tissue homogenates!!
Depends on how the antibody was made. Which strain of mice of used and what was the immunogen. It strikes me as a bit odd that you get a large number of bands from a monoclonal antibody. That means that you either have multiple versions of your target protein or some kind of polyspecific antibody.
our lab has had a long history with this antibody and people tell me that this monoclonal is quite a good one. Ive seen blots on invivo samples using this antibody, which look really good! But, Im not entirely sure of the conditions that were used....reducing/non-reducing....denaturing/non-denaturing?? Neither do I know if something special was done to make this antibody work on mouse samples! I havn't had a lot of luck going through that postdoc's notes...apart from the fact that samples arent supposed to be boiled! Anyways, Im going to try a non-reducing sample buffer without boiling....lets see how it works