John.Kalns at brooks.af.mil (Kalns John E Contr USAFSAM/FEH) wrote:
:We get <1m dots all over films from Western blots run on a Hoeffer system
:(Mighty Small). We do not get this type of background when we run the same
:protocol using a BioRad system. The Biorad system has bigger problems and
:we want to change over to Hoeffer and stop using pre-cast gels. The little
:black dots on the film would be OK if we were only probing for a single
:protein but now we are probing with antibodies against various
:post-translational mods and so pick up lots of bands and want to show the
:whole thing.
Ah, showing a full result is such a rarity these days :-)
:Anybody have a similar experience and find a solution?
:Problems during transfer? Thinking of doing an experiment of running the
:gel on Hoeffer and transfering using Biorad with a good cooling system.
:HELP!!!!
Here is what I concluded about dots problem for myself:
Dots are result of small pieces of gel sticking to the
membrane. Antibody can get trapped in the gel, and then are
washed out poorly (diffusional problems), resulting in
intense localized signal upon development with low MW
substrates. That's why most of the dots are usually
located on the periphery of the blot - where gels
tend to stick most.
Now I inspect blots visually and try to remove any
gel pieces with a jet from squeeze bottle. I think
it helps.
- Dima
