I'm developing a variation of the blue native agarose gel
electrophoresis (BNAGE) method (Henderson et al. 2000. Separation of
intact pyruvate dehydrogenase complex using blue native agarose gel
electrophoresis. Electrophoresis. 21(14):2925-2931.) The original
method is a two-dimensional analysis, wherein the native gel is then cut
into separate lanes and used as the source for the denaturing gel.
Since they're analyzing a heteromeric pyruvate complex, this makes
perfect sense.
I got the two-dimensional version to work, but in my own case, I am
trying to develop a way to study approximate proporations of aggregation
and assembly of viral capsid subunits (55kDa each) into a homologous VLP
of roughly 10MDa (yes, that's megadaltons) in a foreign expression
system.
Since I have no actual need to analytically separate heterologous
subunits from each other, I would like to eliminate the second dimension
and transfer from the agarose gel directly to PVDF for Western analysis.
Breaking up/denaturing the VLPs and other aggregates would not cause me
a problem. I don't need them to be intact on the blot, only to not be
denatured while run on the agarose gel.
I have already tried a simple tank transfer from from the freshly-run
gel, using my running buffer (MOPS-Tricine, pH7, with 0.005%
Coomassie Brilliant Blue G) plus 10% MeOH as the transfer buffer. No
luck.
I now intend to try soaking the gel 30 minutes in two volumes
tris-glycine plus 1% SDS, 1% mercaptoethanol, then soak it another 2x30
minutes in 5 volumes tris-glycine plus 0.1% SDS, 10% MeOH and then try a
tank transfer in the tris-glycine buffer.
Anyone have any suggestions? Should I stick with MOPS-tricine for the
soaks and transfer? Can I just eliminate the BME altogether?
Thanks
Bryan J. Maloney
Boyce Thompson Institute for Plant Research
PS: BNAGE is not a pI-based method. It does not rely upon the
native charge of the protein.
PPS: I'm very familiar with sucrose gradients, but these are cumbersome
and still have to be analyzed at the end via ELISA or Western blot.
Likewise, they are by necessity discontinuous in analysis (even if
continous in creation), and the finer a picture I want, the more
fractions have to be collected. Likewise, I'm limited by the need to
run controls, which means that only a few actual samples can be run on a
single spin.
--
"A 'Cape Cod Salsa' just isn't right."
