I ran 10ug total protein (from RIPA harvest) my lanes and used Kaleidoscope protein standard. I can detect the fluorescence within the Kaleidoscope standard at the low molecular weights, and the colors from the standard are very clear on the PVDF membrane, suggesting a good transfer. However, I am unable to detect GAPDH using a anti-GAPDH (Cell Signaling) antibody @ 1:1000 and Invitrogens Alexa488 secondary @ 1:2000. I know fluorscence is inferior to chemiluminscence in terms of amplifying a signal, but 10ug protein should be sufficient to see a band.
When I place my western on a blue light transilluminator I can clearly see the fluorescence from the standard; however, there is no signal in the body of the blot. I attempted to detect the bands using an EpiBlue filter on a gel imaging system using 100ms exposure. Is this long enough?
Edited by Ahrenhase, 21 October 2014 - 08:38 PM.