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 
perfect sense.

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 
single spin.

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 
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


Please note:  BNAGE is not a pI-based method.  It does not rely upon the 
native charge of the protein.




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