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Visualising Large Proteins

Ellen K. LeMosy ellen.lemosy at yale.edu
Thu Sep 12 10:04:06 EST 1996

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 

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