pre stained marker issues - (Mar/08/2010 )
I am running a 5% gel and am seeing some issues- I transfected HEK293 cells and fibroblasts with my plasmid of interest. I obtained the cell lysates by incubating the cells with RIPA buffer, centrifugation and collecting supernatant.
When I run the gel, the pre stained marker is not separating at all. My protein size is 110KD. I only see the blue line from the samples on the gel but no clean separation of the marker.
Can anybody help me on this issue?
Marker hasn't degraded has it? How much did you load? Have you used it before?
a 5% gel is neither very selective for a 110kDa protein nor for your markers.
you should use at least 7.5% but 10% would be better. a gradient (we like 5-15%) might be even better.