EMSA - protein-probe complex stuck in wells? (Aug/07/2011 )
I recently started to run EMSA gels with C. elegans nuclear extracts. However, it seems like or I suspect that the formed protein-probe complexes get stuck in the wells and only very little of the complex migrates into the gel (see attached gel). I load 50ug of total protein on a 4% gel, run in 0.25xTBE for 2hrs at room temp. I prerun the gels and also clean the wells before loading with a syringe. Does anyone know how to prevent getting the complexes stuck in the wells?
There are several reasons why this may happen.
1) too much protein used.
2) the protein of interest aggregates retaining the probe before it enters the gel at a very high molecular weight.
3) the protein/complex is not aggregated but still too large
I would troubleshoot first by decreasing the amount of protein in the probe. You might be interested in checking this EMSA troubleshooting table http://www.nature.com/nprot/journal/v2/n8/fig_tab/nprot.2007.249_T5.html