I ran a Western with several stable clones I had selected and probed for gapdh (had had trouble detecting protein of interest but that's a different story). After a 2 minute exposure two of my clones showed strong staining (these were on the RHS of the gel), I then exposed the same membrane for 15 minutes and these two bands disappeared and the rest of my clones appeared! Nothing was changed during this time, all I did was remove the original film and place the second film on top. After this I washed the membrane, re applied the chemiluminescent reagents and repeated the exposures and had the same thing occur, these two bands appear and then disappear.
Details about the Western:
~15ug DNA loaded
Ran for ~2 hours at 120V
Transfered O/N at 40V at 4 degrees.
Blocked in 10% milk in PBST for 1 hour (shaking)
Primary and secondary ABs incubated for 1.5 hours with 3x 5 mins PBST washes in between, both concentrations 1:2000
Applied chemiluminscent reagents and developed as described above.
At the moment I'm just planning on running a fresh Western and seeing if I can get more normal results..
Edited by -Fran-, 09 August 2011 - 09:51 PM.