Protein transfer from agarose gel.
Bryan J. Maloney
bjm10 at cornell.edu
Tue Jun 26 15:17:45 EST 2001
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
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.
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
I have no actual need to separate 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 gel.
I got the two-dimensional version to work.
I have already tried simply trying a 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
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?
Bryan J. Maloney
Boyce Thompson Institute for Plant Research
Please note: BNAGE is not a pI-based method. It does not rely upon the
native charge of the protein.
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