EMSA - Protein-oligo complex is stuck (Dec/25/2010 )
I'm a new in EMSA and trying to investigate the interaction of NFkB with its consensus after treatment with certain molecule of interest. I'm using non radioactive fluorescent EMSA protocol (our institution is lucky to have Odyssey equipment for this purposes). I've been using The Thermo Scientific NE-PER Nuclear and Cytoplasmic Extraction Kit for nuclear proteins isolation and managed to get about 0.5ug/uL from 5 millions of microglia cells. My problem is that the after 1 hr of running the PA native gel at 80V i'm getting very "lush" signal wich is concentrated at the bottom of the wells. The free probe is running properly and detectable at the bottom of the gel. It's noticeable that there is less amount of free probe at the wells with complex compared to lane with a free probe, but the whole coplex is hardly (if ever) enter the gel. What could be a reason for the stacking complex? I'm using 4% PA gel on TAE buffer basis and running it 1hr at 80 V at TAE buffer. My binding conditions include NP40, DDT and i'm using 5ug of nuclear extract per reaction.
Thanks at advance!
Nobody has any idea?
It would be easier to understand if you could do a competition within your assay. It is a little hard do understand what is happening in your gel this way...
You might be having a supershift in all the wells (the complex Factor-consensus sequence is to big to get in the gel).