I'm gearing up to do some microsequencing of proteins detected
by SDS-PAGE and am looking for some advice:
Is a Tris-Tricine buffer system better to use than the standard
Tris-Glycine? I read that this may prevent N terminal blocking.
Has anyone experience with both systems prefer either one? My
protein bands appear a bit diffuse. How do I tighten them up?
I'm using a Bio-Rad Mini Protean II Trans-blot Transfer Cell
and am considering using Millipore Immobilon-P PVDF membranes
for the support matrix. Bio-Rad claims that their PVDF membranes
are superior to the competition because they retain the proteins
much better during the transfer, ie. a second membrane placed
behind the first reveals less protein seepage. Which membrane
works better in your hands? Has anyone worked out the best
conditions for electroblotting using the above membranes, buffers,
and electrophoresis unit?
I would like to stain the gel with a copper stain kit (it's
supposed to be more sensitive than Coomassie Brilliant Blue
R-250). Has anyone tried this stain kit? Is it really much
more sensitive? What's the difference between CBB G-250 and
R-250? What does the G and R stand for? Is it best not to try
and visualize the proteins before transfer, and then stain the
membrane with CBB? Will this work for the copper stain as well?
Paul N. Hengen
National Cancer Institute
Frederick Cancer Research and Development Center
Frederick, Maryland 21702-1201 USA