Hi. My experience is with extracellular matrix proteins, which are also
large. Regarding gels, a 5% SDS-PAGE gel will give good resolution in
the 150-400+ kDa range; a 4-12% gradient gel will expand that to about
70-400 kDa if you need to see smaller marker proteins as well; both of
these are run with a 3% stacking gel. Very good transfer of your protein
out of these gels can be achieved using semi-dry blotting. I use the
Pharmacia NovaBlot apparatus; while its graphite plates eventually wear
out, it does not heat up (and warp) with the extended transfer times
required for large proteins. The formula for transfer is 2 hours at
constant milliamps = 0.8 x (surface area of gel in cemtimeters). For an
8 x 10 cm minigel, this is 64 mA. Convenient markers are things like
matrix proteins (fibronectin - 220 kDa, human tenascin- 320 kDa; also
laminins, fibrinogen) which are commercially available somewhere.
Thyroglobulin is also large....
Good luck. It is certainly possible to do gel and blot analysis of a 290
kDa protein! Ellen
