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Western: Mouse antibody on mouse tissue homogenates

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3 replies to this topic

#1 Sora



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Posted 02 April 2011 - 08:02 PM

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!!

#2 BioMiha



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Posted 02 April 2011 - 11:54 PM

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.

#3 Sora



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Posted 04 April 2011 - 04:16 PM

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

#4 proteaMatt



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Posted 05 April 2011 - 06:08 AM

Someone on these forums (can't remember the topic offhand) had a similar issue with mouse tissue. Their problem turned out to be endogenous immunoglobins (i'm pretty sure it was IgE) in the tissue sample itself.

Here is the link to that topic, that person never responded to the topic to report if they found anything that helped them.

Edited by proteaMatt, 05 April 2011 - 06:12 AM.

Lab Technician at Protea Biosciences

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