EMSA - probe disappears with nuclear extract - probe works without nuclear extract... (Mar/08/2011 )

Hello,

Please find attached my blot for the EMSA. The left lane is control sample and the lane on the right is serum treated mesangial cells. For some reason, when I incubate my AP-1 probe with nuclear extracts, I do not pick up the free probe. The blot in the left corner is where I loaded my control sample. Does anyone have an idea of what might be the problem? My possible reasons are that the samples did not enter the gel, the loading buffer from the kit degraded the probe, or that the temperature of the gel was too high. For my gel, I pre-electrophoresed for 1 hour at 100 V and ran the samples at 100 V for 1 hour and 40 min.

Thank you

too much protein?