Visualising Large Proteins

Dpt. of Cell Morphology Cell.Morphology at rz.ruhr-uni-bochum.de
Fri Sep 13 11:42:17 EST 1996


Dear Mr. Doog,


I have no experience with large proteins like that, but can't resist to 
give you some tips:

Tatsumi and Hattori resolved Myofibrillar Proteins of 3000 - 700 kDa with 
the Help of a 2% Polyacrylamide Gel strengthend with Agarose. You find 
sufficent instructions in their publication in Analytical 
Biochemistry,244,28-31 (1995) (You may e-mail me and I'd send you the 
article as a Fax, if you like.)

A more conventional method for the separation of large proteins would be 
to pour a gradient gel, perhaps a 5 to 10 % gradient should work with 
your 290 kDa Protein and can be handeled without too much trouble.

For western blotting of large proteins, you should omit methanol from the 
buffers to allow swelling of the gel.


> unable to confirm the size of bands which I see. Bands which may indicate
> that expression is occuring remain in the stacking gel.

I've never resolved anything larger than conventional 205 kDa Myosin, but 
that dind'nt make any problems using a simple 10 or 7,5 % PAGE-gel. I 
consider it unlikely, that 290 kDa Proteins stay in the stacking gel in a 
7,5% gel.

Good Luck

Ralf Breuker, at the
Department of Cell Morphology
NDEF 05 / Ruhr University Bochum
44780 Bochum, FRG



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