1 reply to this topic

[mastermi]

Hi all,

I would like to make a North-Western Blot to detect RNA-protein interaction.
Is there something special I have to consider?
Or can I simply use a standard protocol for Western Blot hybridization and detection?
I am making a vacuum RNA blot on a nitrocellulose membrane. I would then incubate the fixed membrane with cell extract and afterwards start the standard protocol with my first antibody...

Thanks in advance!

Edited by mastermi, 11 June 2009 - 06:01 AM.

[drjcroberts]

hi,

north westerns are really frought with problems, you will get lots of background and very little signal unless you are looking at something that is very abundant in the cells.

a better approach is to use RNA-ChIP, also known as CLIP assays. this uses the same principles as chromatin immunopreipitation but isolates RNA-protein complexes. i have some protocols if you are interested. you can then detect your sequences with a standard northern after IP or with RT-PCR
\J