urbig at biokemi.su.se
Mon Nov 17 04:45:24 EST 1997
In article <64equa$8e0_001 at leeds.ac.uk>, BMBLDO at leeds.ac.uk (L.D. Ofner) wrote:
> Dear all,
> I've been trying to run a 280 KDa protein on a native polyacrylamide gel and
> have had problems getting it to actually enter the separating gel. The
> protein comprises of two 140 KDa subunits that I have digested with trypsin
> to yield 140, 94 and 45 KDa fragments which I have then subjected to native
> page. I can't use SDS as I am looking at the activity of these fragments in a
> gel overlay experiment. I have tried altering the % of the gel but to no
> avail; I still get squished bands at the very top of the separating gel that
> has barely left the stacking. I've heard that agarose can be used in a native
> gel to overcome problems with large proteins not entering (although I didn't
> think my protein was that big to cause such problems!)and if so can anybody
> send me a protocol.I would be extremely grateful for any other suggestions.
> Lisa (bmbldo at leeds.ac.uk)
Try the following continuous gel system (no separate stacking gel, keep
sample volume as small as possible):
Acrylamide (30/0.8 Bis) final 6% (use a thicker gel, possibly 3mm)
Buffer final 100-150 mM
Saccharose (70%stock) final 7-21% (depending on your problem)
TEMED 13 mikrol/10ml
10%APS 40 mikrol/10ml
as buffer use 1M Tris/0.73M boric acid, pH should be around 8.6,
dilute this buffer 1:10 as running buffer.
I also tried the "normal" Tris/Glycine (30g/l-144g/l) system as continuous
gel system for native gels, no separate stacking gel, running buffer is
1:10 diluted, same for the gel. As a rod gel (about 30 mm diameter) I used
this down to 3% Acrylamide-concentration.
Both buffers work also with agarose, start at about 2-2.5% agarose(fine
for preparative purpose in rod gels).
email: urbig at biokemi.su.se
More information about the Methods