Dr. Frank O. Fackelmayer
fof1 at chclu.chemie.uni-konstanz.de
Fri Mar 14 10:56:41 EST 1997
In article <5g7n7v$3d8$1 at scream.auckland.ac.nz>,
mmm_klehnert at mednov1.auckland.ac.nz (KLAUS LEHNERT) wrote:
> Hi netter's
> does anyone out there have a good (and tried) protocol for
> Tris-Tricine Polyacrylamide gels?
> I have tried the protocols in the "original" publication
> (Schaeger+Jagow 1987), I'm using high-quality reagents, but at
> the very best I'm getting very fuzzy bands, and even when I run
> the gel (in a BioRad Mini-ProteanII) for twice as long (i.e.
> duoble the time the bluemarker needs to run out), my smallest
> marker band (Aprotinin, 6.5kDa) is still in the upper half of
> the gel.
> Any suggestions?
I also had some problems with the original protocol of Schaegger. I´ve
changed some things and now it works like a charm.
1. Always run 12%gels (not 16.5% or with two layers of resolving gel)
2. Don´t use any glycerol in the gel (replace its volume by water)
3. Use a minigel (some 6cm high), not a long gel !!!!!
(From what I´ve learned while optimizing the procedure, this is the most
important step. Although your bands are quite close to each other, the
separation is great, at least down to 3kD. Using longer gels does not only
produce fuzzy bands, but also takes an awful lot of time.)
4. Run your minigel at low current (30V/approx. 15mAmps) until samples are
in the stacking gel, than change to higher current (say 30mAmps) and run
your gel until the coomassie blue just runs off the gel. In my case, this
takes some 60min, but of course it is dependent on your gel dimensions.
Try varying your electric parameters to have the gel finished in one hour.
5. Stain your gel with fixating Coomassie Blue (Steck et al. 1980, Anal.
Biochem. 107:21-24 is great for that).
If the problems remain, change your acrylamide solution.
Hope this helps,
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