I have been running lots of western lately with no problems....until now.
It is time to re-run blots to get "the pretty picture", so I am using the same samples, membranes, primary, secondary, buffers, detection reagent, etc. When I develop, I usually exposure for 30 seconds to make sure things are straight, then 3 minutes, then 10 minutes (my protein is not so abundant). Generally, I will only see my standard (MagicMark) at 30s, faint bands at 3min, and pretty nice bands at 10min. Now, I barely see the standard at 30s, at 3min I see the standard OK, and see my bands faintly, but at 10 minutes I see nothing. It isnít background blown out--it is just all white--you can see the outline of the membrane if you crank up the gamma. But, no bands are visible, no sample bands and no standards. I originally thought that our imager must be broken, but no one else in the lab is having this problem. I have even borrowed their blots and exposed them repeatedly, and their bands only get darker with increased exposure time. What is going on with my sample?? Any ideas on why the bands would completely disappear between a 3min exposure and a 10min one?