North-Western Blot - (Jun/11/2009 )
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!
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
Can I get those CLIP protocols please? If you also tried a western blot which worked, could you please send that protocols to me aswell? Thank you very muchª!