An annoying Western blotting problem - (Mar/21/2006 )

please see the attachment.

in my study, the protein of interest is around 50kDa. however, i don't know why i always get a blank (or hollow) on the blots (PVDF/ AP-BCIP/NBT)? it seems that the protein of interest was entirely "removed" from the blot. i re-did it with 10X dilute antigen sample and 10x dilute primary Ab, respectively, but the annoying problem still remains unsolved.

who can tell me what i should do? my email: qcsnowman@hotmail.com

thanks in advance for your valuable suggestion.

any idea?

you are seeing a 'ghost band'

search this forum for 'ghost band' and you'll get some good stuff, it comes up somewhat often and you can get good suggestions that way.

thanks aimikins for your reply.

i have searched this forum as you suggested.

in my case, i am using secondary Ab conjugated with AP (BCIP-NBT). but i encountered the same "white-band" problem as described by others who used HRP/ECL system. so it seems to be least to support that "white band" is formed due to the presence of too much HRP on the blots.

some blots on the same membrane were always stained clearly, eg. the band nearly 30kDa (see my photo), irrespective of whatever dilutions of primary or secondary Ab (at 1:5k, 1:10k, 1:20k, or 1:40k) i tried. but why the 50kDa-band seems to be so resistant to the "staining" ?