I'm running 2D gels then transferring to PVDF membrane. Up until now no problems.
This recent batch however I'm having issues with.
The first issue the ladder transferred perfectly but no protein transferred to membrane (there was 200 ug protein total on the 2D gel). Why would the ladder transfer and not the proteins? Ponceau on the blot revealed no spots and when I coomassie stain the gel there are some spots still on the gel but not many, suggesting they should have transferred.
The second issue is this weird horizontal line in the blot when I develop it (see attached images). It's not a "streak" from the 2D gel. It's something else that I haven't seen before and its not appearing in the coomassie stained gel - just the transferred blot. What could it be?