native gel of large complexes

[Jeffrey W. Chisholm]

In article <3kitg3$8c4 at news.ycc.yale.edu>, chamo says...
>
>I'm trying to do a native gel of a large protein complex (>550 kDa), 
but I'm
>having trouble getting it into the gel.  Has anyone tried to use 
agarose intead
>of acrylamide?  Has this also worked for gel shifts?  Any and all 
advice will
>be greatly appreciated.
>
>Danny
>
Danny,

Your question is difficult to answer without more detail and I can't 
reply in person without your email address as <chamo> is not a full 
internet address.  What percentages of acrylamide have you tried, and 
how much greater than 550 kd is your complex?  Try making a 3-10% gel 
with a 2.5 percent stacker if necessary for your electrophoresis 
apparatus and run it in Tris-Borate buffer pH 8.3 at 10 C for 24 hrs 
(to equilibrium) at around 200 volts.  Gels of this type work for my 
analysis of lipoproteins (much greater than 550 kd) with a 
Thyroglobulin Std. (669 Kd) migrating well into the gel.  In practice, 
I have been able to polymerize acrylamide at 2% under a t-butanol 
overlayer and to 2.3 percent without oxygen-free conditions.
  I may be able to give you more info if you tell me what you are 
trying to do and what equipment you have to work with.  As for 
agarose, it might work in a 2 or 2.5% gel provided you don't need 
great resolution and have time to play with conditions.  BTW have you 
tried running you protein native on a biorad mini-gel system with 
Biorad precast 4-20 Tris gels as proteins up to about 600-650 Kd 
should enter.

Jeffrey W. Chisholm
Lipoprotein Research Group
Dept. of Biochemistry
Dalhousie University
Halifax, Nova Scotia, Can.

Email: JWChish at ac.dal.ca