We have a lab with not much experience with western blot and trying to set it up working. It goes OK most of the time and have a working system. However recently I had extremely narrow bands when I stain my membrane with Ponceau see i46.tinypic .com /29779h.jpg
with the exception of the last but one lane. I had an indentical problem running these samples previously and had a crappy western blot result (read black lane).
I use precast gels from the NuPage system, which should rule out errors on the gel part since I used this batch succesfully. These samples are HELA cells lysed directly with sample buffer, stored in -80 and pipetted up and down in a high gauge needle several times.
Possible causes I am considering is too little shredding, running too long at a low voltage and hence the overheating (would not explain the single correct lane), possibly a salt concentration problem and this single sample would be just ok. (on the previous run it had the same appearance as the other lanes).
I am pretty much at a loss what this can have been, any ideas?